Antibody selective for a tumor necrosis factor-related apoptosis-inducing ligand receptor and uses thereof

ABSTRACT

An antibody of the invention interacts with human DR5 or with human DR4 to produce agonistic or antagonistic effects downstream of the receptor including inhibition of cell proliferation and apoptosis. Nucleic acid sequences and amino acid sequences of DR5 and DR4 antibodies have been elucidated and vectors and cells containing and expressing these sequences have been generated. Methods and uses for the antibodies are detailed including treatment of apoptosis-related disease and treatment of dysregulated cell growth.

ACKNOWLEDGEMENTS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/346,402, filed on Nov. 1, 2001, and claims priorityto PCT/US01/14151, filed May 2, 2001, which is currently pending.PCT/US01/14151 claims the benefit of U.S. Provisional Application No.60/201,344, filed May 2, 2000. The applications to which the presentapplication claims benefit are herein incorporated by reference in theirentirety.

[0002] This invention was made with government support under Grant NCIP50 CA 89019-01 awarded by the National Cancer Institute and under NIHR03-AR44982 awarded by the National Institute of Arthritis andMusculoskeletal and Skin Diseases. The government has certain rights inthe invention.

FIELD OF THE INVENTION

[0003] The present invention relates to an antibody capable ofspecifically binding a single type of tumor necrosis factor (hereinafterreferred to as “TNF”)-related apoptosis-inducing ligand (hereinafterreferred to as “TRAIL”) receptor, more particularly, to a monoclonalantibody that induces apoptosis in in vivo and in vitro cells expressingthe single type receptor and therapies based thereon.

BACKGROUND OF THE INVENTION

[0004] TRAIL is a member of the TNF family of proteins, which alsoincludes TNF-α and Fas ligand (1). These proteins are potent inducers ofapoptosis. To date, five receptors for TRAIL have been identified, twoof which, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (2-7), are capable oftransducing the apoptosis signal while the other three DcR1 (TRAIL-R3),DcR2 (TRAIL-R4), and osteoprotegerin (OPG) do not transduce theapoptosis signal (8-12). All five receptors for TRAIL share significanthomology in their extracellular ligand binding domains. Similar to Fasand TNF receptor I (hereinafter referred to as “TNFRI”), theintracellular segments of both DR4 and DR5 contain a death domain, andtransduce an apoptosis signal through a pathway that involves theFas-associated death domain protein (hereinafter referred to as “FADD”)and caspase 8 (6,7). In addition to transducing the apoptosis signal,the DR4 and DR5 receptors can also activate a pathway involving NFκb(6,7).

[0005] The biological functions of TRAIL that have been demonstratedinclude the capability of TRAIL to selectively induce apoptosis oftransformed tumor cells, with normal cells being relatively resistant toTRAIL-mediated apoptosis (13-15). This selectivity suggests that, incontrast to Fas ligand, the administration of TRAIL is associated withvery low levels of toxicity as demonstrated by systemic administrationof TRAIL in an animal model without inducing significant toxicity (13).Thus, TRAIL has been proposed as a potent apoptosis inducing agent thatwould be a suitable therapeutic agent for the treatment of cancer andother diseases associated with abnormal cell proliferation. TRAIL alsohas been proposed to be a potent apoptosis-inducing agent that would besuitable for the treatment of autoimmune and inflammatory diseases. Ithas been demonstrated that TRAIL-mediated apoptosis is involved inactivation-induced cell death of T cells, thereby serving as analternative mechanism to Fas ligand (16,17). TRAIL-mediated apoptosismay also function in the induction of apoptosis of T cells and otherinflammatory cells (18), and plays a role in the killing activity of NKcells (19-21), and in the immunomodulatory function of dendritic cells(22,23). Thus, TRAIL-mediated apoptosis may also function inimmunoprivilege and immunosurveillance.

[0006] The TRAIL receptor system is complex, and includes at least twodeath receptors, DR4 and DR5, and at least two non-apoptotic receptors,DcR1 and DcR2. All of these receptors not only share a high amino acidsequence homology, but also exhibit a similar binding affinity to TRAIL(2-12). The ability of the DcR1 and DcR2 receptors to compete forbinding of TRAIL without inducing apoptosis suggests that they may actas decoy receptors that block or modulate the activity of the TRAILligand. Moreover, it has been reported that untransformed cells expresshigher levels of decoy receptors than do transformed cells. Thus, it hasbeen proposed that the differential modulation of the expression of thedeath and decoy receptors may represent a key regulatory mechanism thatdetermines the susceptibility of cells to TRAIL-mediated apoptosis, butdue to the lack of receptor-specific antibodies (2). Although theexpression and function of DR4 and DR5 have been studied extensively,progress has been impeded by the lack of receptor-specific monoclonalantibodies. The cell surface expression of DR5 has not been documented.It has been reported that a panel of anti-TRAIL receptor antibodies havebeen generated that are capable of inducing apoptosis of melanoma cellsin vitro but only upon immobilization of the antibodies, to promotecross-linking, and, in some cases, the cells require culturing withactinomycin D (24). Several anti-DR5 antibodies have been generated(24). However, these previously generated anti-DR5 monoclonal antibodieshave low apoptosis-inducing activity in vitro, even under the conditionsof crosslinking. No in vivo activity has been reported. These antibodieshave not been used for examining cell surface expression of TRAILreceptors (24). Thus, there exists a need for a monoclonal antibodyselective for each specific TRAIL receptor that is not only able to bindto cell surface receptor but also to strongly induce apoptosis ofvarious types of abnormal cells, including tumor cells, both in vivo andvitro without the requirement for crosslinking or immobilization. Suchan antibody would not only provide potential therapeutic agent but alsoa diagnostic tool for functional analysis of TRAIL receptor. Thereexists a particular need for an antibody specific against each of thedeath inducing receptors DR4 and DR5.

[0007] In the development, or progression, of many diseases it is oftenthe case that cells are not deleted. In many autoimmune diseases andinflammatory conditions, the surviving activated cells attack normaltissues or cells. Further, progression of tumorigenesis and theproliferative panus formation of rheumatoid arthritis are characterizedby the unchecked proliferation of cells. Thus, insufficient apoptosisleads to the development of disease, and the uses of apoptosis-inducingligand or agonistic monoclonal antibody to enhance apoptosis areconsidered as a potential therapeutic strategy for eliminating thoseunwanted cells.

[0008] For example, rheumatoid arthritis (hereinafter referred to as“RA”) is a common human autoimmune disease. The current understanding ofthe pathophysiology of RA is that autoimmune T cells and B cellsinitiate an inflammatory response in the joints, which driveshyperproliferation of the synoviocytes. As a consequence of thehyperproliferation of synovial cells, metalloproteinases (hereinafterreferred to as “MMPs”) are over-produced, which further leads to theerosive destruction of the cartilage and bone that is characteristic ofRA (25). Thus, the control of hyperproliferation of inflammatorysynovial cells is a key step in the treatment of RA. The molecularmechanisms leading to the hyperproliferation of synovial cells are stillunknown. Although the hyperproliferative synovial cells arenon-malignant and non-transformed, many studies have suggested that theyshare some common features with transformed cells (46). These cells, theso-called, “transformed-appearing synoviocytes”, are characterized by adense rough endoplasmic reticulum, numerous irregular nuclei, andchanges in the normally spindle-shaped cell skeleton. It has beenproposed that the incorporation of the oncogenes and virus-derived genesmight be the primary triggers for the transformed appearance of RAsynovial cells (46).

[0009] At least two aspects of RA suggest that dysregulated apoptosismay contribute to the disease process and that therapeutic elicitationof apoptosis may be an effective treatment: the failure of the deletionof the activated T cells suggests that there is defectiveactivation-induced cell death of these T cells, which is a process thatinvolves Fas-mediated apoptosis and TRAIL-mediated apoptosis, and thehyperproliferative nature of the RA synovial cells is a contributingfactor in the later stages of RA pathophysiology. Indeed, it has beenshown that the administration of anti-Fas antibody into the inflammatoryjoint inhibits the development of chronic arthritis in tax transgenicmice, which are an animal model for human RA (26). Moreover, localizedtransduction with the fas ligand gene by an adenoviral vector iseffective in prevention of collagen-induced arthritis (27). Inhibitionof the proliferation of inflammatory synovial cells by enhancement ofFas-mediated apoptosis is observed in both cases. Although Fas ligand isa strong apoptosis inducer in RA synovial cells, the application of Fasligand-mediated apoptosis as a therapy for humans has been limited bylethal liver toxicity. Thus, TRAIL receptor induced apoptosis representsa safer and more effective therapeutic for the treatment of RA thanFas-ligand induced apoptosis. TRAIL receptor induced apoptosis alsorepresents a safer and more effective therapeutic for the treatment ofcancer than Fas-ligand induced apoptosis. TRAIL-mediated apoptosis isknown to specifically induce apoptosis of transformed tumor cellswithout affecting normal cells. It has been shown that the systemicadministration of the trimerized soluble TRAIL did not cause toxicity inexperimental animals yet was able to induce regression of implantedtumors (13,28). Its potential as an adjunctive therapy for traditionaltreatments was underscored by the recent finding that the expression ofDR5 and susceptibility to TRAIL-induced apoptosis of breast cancer cellsis enhanced by the radiation, suggesting that combined with radiation,the efficiency of TRAIL would be increased in cancer therapy (29).

[0010] In addition, the gene encoding the TRAIL receptor DR5 has beenmapped to chromosome 8p21-22, loci with a high frequency of mutation insome cancer cells (30). It has been reported that at least two kinds oftumor cells, small lung cancer (31) and head and neck cancer (32)exhibit mutations in the death domain of the DR5 gene. Thus, thereexists a need for an anti-DR5 antibody in cancer research to determinethe effect of receptor epitope variation on the development andprogression of cancers. Further, the functionality of TRAIL receptormutations would prove a useful clinical diagnostic tool when used inconjunction with other biomarkers in the early detection of cancers andas a predictor of the tumor aggressiveness.

SUMMARY OF THE INVENTION

[0011] In one embodiment, the invention relates to an antibody whichrecognizes a TRAIL receptor DR5 and which induces apoptosis in aDR5-expressing cell in vivo or in vitro. Further disclosed is anantibody that recognizes DR5 but not DR4, DcR1, or DcR2. Specificallydetailed is a monoclonal antibody to DR5 produced by a hybridoma.

[0012] In another embodiment, the invention relates to an antibody whichrecognizes a TRAIL receptor DR4 and which induces apoptosis in aDR4-expressing cell in vivo or in vitro. Further disclosed is anantibody that recognizes DR4 but not DR5, DcR1, or DcR2. Specificallydetailed is a monoclonal antibody to DR4 produced by a hybridoma.

[0013] A method provided is induction of apoptosis in target cells orinhibition of target cell proliferation by contacting a cell with atherapeutic quantity of an antibody capable of binding to DR5 or DR4. Invarious embodiments of the method, the apoptosis can be induced or thecell proliferation inhibited by contacting the target cells with bothantibodies.

[0014] Also disclosed is a pharmacological composition that includes atherapeutic amount of monoclonal antibody active against a DR5 or DR4, apharmaceutically acceptable carrier and, optionally, a containerenclosing the antibody and the cancer. Further provided by the inventionis the use of an antibody recognizing DR5 or an antibody recognizing DR4for preparing a therapeutic for selective apoptosis of abnormal ordysregulated cells.

[0015] An antibody of the present invention interacts with a tumornecrosis factor related apoptosis-inducing ligand receptor such as DR4,DR5, DrR1, DrR2 and OPG, inducing apoptosis in a cell expressing such areceptor. Disclosed is an antibody of the invention capable ofselectively binding an agonistic or antagonistic tumor necrosis factorligand receptor epitope.

[0016] The present invention provides a treatment for an apoptosisrelated disease, cancer, inflammatory disease, or an autoimmune diseaseby a method that includes contacting a target tissue having the diseaseto a therapeutic quantity of an antibody of the invention, singly or incombination with other apoptosis inducing antibodies, and/or othertherapeutic agents or treatments.

[0017] Further described is a fusion protein that includes an antigenicTRAIL receptor amino acid sequence having at least ten bases, coupled toan immunoglobulin protein or fragment thereof capable of eliciting animmune response within a subject.

[0018] The present invention provides a method of gene therapy in whicha target cell is transfected with a TRAIL receptor nucleic acid sequencein an expression vector so that the TRAIL receptor is expressed on thetarget cell. The target cell is then exposed to an antibody thatselectively binds the TRAIL receptor.

[0019] Provided are nucleic acid sequences and amino acid sequencesencoding the heavy and light chain immunoglobulins of an antibodyselective for DR5. Sequences are also provided for an antibody thatselectively binds DR4. Also detailed are vectors that include a nucleicacid sequence of the invention and host cells transformed with a vectorof the invention.

[0020] The present invention provides a humanized DR5 antibody (e.g.,TRA-8) and a humanized DR4 (e.g., 2E12), as well as a transfected cellproducing the humanized DR5 antibody and a transfected cell producingthe humanized DR4 antibody.

[0021] A process for producing a humanized DR5 antibody or DR4 antibodyis described in which a host is transformed with nucleic acid sequencesencoding a humanized immunoglobulin light chain and a humanizedimmunoglobulin heavy chain after which the transformed host is incubatedfor a predetermined period of time.

[0022] Also described is a process for inducing apoptosis in targetcells or for inhibiting cell proliferation that includes contacting atarget cell with a pharmaceutically effective amount of a humanized DR5antibody, a humanized DR4 antibody, or a combination of both, in thepresence or absence of other therapeutic agents and treatments.

[0023] A commercial kit is provided for inducing apoptosis that includesa humanized TRA-8 antibody selective for DR5 or a humanized antibody forDR4 (e.g., humanized 2E12), packaged in a suitable container, andoptionally with instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1. Characterization of TRA-8. (a.) Binding specificity ofTRA-8: Western blot analysis (upper panel): Recombinant fusion proteinsof the TNFR family probed with TRA-8 or anti-human IgG. Lane 1:DR5/hIgG1 fusion protein (immunogen); Lane 2: DR4/hIgG1 (TRAIL-R1); Lane3: DR5/hIgG1; Lane 4: TRAIL-R3 (DcR-1)/hIgG1; Lane 5: TRAIL-R4(DcR-2)/hIgG1; Lane 6, CD95/hIgG1; Lane 7: soluble TNFRI. ELISA analysis(lower panel): The well numbers match those of the Western blot exceptwell 8 which is a murine DR5/hIgG1 fusion protein. (b.) Binding activityof soluble TRAIL and TRA-8 to DR5 and DR4: ELISA plates were coated withDR5/hIgG1 (left panel) or DR4/hIgG1 (middle panel) and then incubatedwith TRAIL or TRA-8. (c.) Flow cytometry analysis of the surfaceexpression of DR5. Cos-7 cells transfected with pcDNA3 expression vectorcontaining the full-length DR5 cDNA (solid histogram), DR4 cDNA (openhistogram, solid line) or empty vector (open histogram, dashed line).Forty-eight hours after transfection, cells were stained with TRA-8followed by PE-conjugated anti-mouse IgG1. (d.) in situimmunohistochemistry reactivity for DR5: Cytospin slides of Cos-7 cellstransfected with DR5 expression or control vector were stained withTRA-8 at 48 hours after transfection, (e.) Killing activity of TRA-8:Jurkat cells were incubated with the indicated concentrations of TRA-8.Cell viability was determined by ATPLite, MTT, and PI exclusion assaysafter overnight culture. The results of ATPLite and MTT assays arepresented as percent of medium control, and PI assay are presented aspercent of PI negative cells (f.) Western blot analysis of caspaseactivation: Jurkat cells were incubated with 500 ng/ml TRA-8 forindicated time. Cell lysaies were separated by 15% SDS-PAGE, blotted,and probed with anti-caspase antibodies. The arrows indicate the cleavedsubunits of each caspase. g. Caspase inhibition assay: Jurkat cells wereincubated with 50 ng/ml TRA-8 overnight in the presence of variousconcentrations of indicated caspase inhibitors. Cell viability wasdetermined by the ATPLite assay.

[0025]FIG. 2. Cell surface expression of DR5 and susceptibility toDR5-mediated apoptosis. Normal T and B cells, freshly isolated fromperipheral blood, T cell (a and a′), glioma (b and b′), prostate cancercell (c) and B cell (d) cell lines were incubated with TRA-8 or murineIgG1 isotype control antibody followed by PE-conjugated goat anti-mouseIgG1. The open histograms represent the isotype antibody control whilethe solid histograms represent TRA-8 staining. Apoptosis was determinedby the ATPLite assay after overnight incubation with soluble TRAIL (opencircles) or TRA-8 (closed circles) as shown in a, b′ and d.

[0026]FIG. 3a′ T cell line U937 was incubated with TRA-8 or murine IgG1isotype control antibody. Apoptosis was determined by the ATPLite assayafter overnight incubation with soluble TRAIL (open circles) or TRA-8(closed circles).

[0027]FIG. 3 Glioma (b) and prostate cancer (c) cell lines wereincubated with TRA-8 or murine IgG1 isotype control antibody. Apoptosiswas determined by the ATPLite assay after overnight incubation withsoluble TRAIL (open circles) or TRA-8 (closed circles)

[0028]FIG. 4 is a series of graphs showing cell viability for humanJurkat cells after exposure to indicated concentrations of (A) antibodystrains TRA-1, -8 and -10 and (B) TRAIL in the presence of a fixedconcentration of the inventive antibody strains depicted in FIG. 4A;

[0029]FIG. 5. Expression of DR5 in normal and cancer tissues: Normal andcancer tissue homogenates were probed with TRA-8 and developed bychemiluminescence. (a.) Western blot analysis of DR5 protein in normaltissues: lane 1: liver, lane 2: brain, lane 3: lung, lane 4: kidney,lane 5: spleen, lane 6: testes. Lane 7: ovary, lane 8: heart, lane 9:pancreas. b. Western blot analysis of DR5 protein in cancer tissues. Thecancer tissue blot containing cancers from the ovary (lane 1), lung(lane 2), liver (lane 3), rectum (lane 4), cervix (lane 5), skin (lane6), testes (lane 7), thyroid (lane 8), uterus (lane 10), stomach (lane11), laryngopharynx (lane 12), and pancreas (lane 13) was probed. Insitu immunohistochemistry of normal human tissues (c.) and of cancertissues (d.). Frozen sections were immunostained with TRA-8.

[0030]FIG. 6. Tumoricidal activity of TRA-8. SCID mice were inoculatedsubcutaneously with 1321N1 cells. Mice were injected intravenously witha single dose of 100 μg TRA-8 on the second day after tumor inoculation(a.), or with three doses of 100 μg TRA-8 beginning 7 days after tumorinoculation (b) Tumor growth was determined by the weight and examinedhistologically with H&E staining. The photographs show viable tumorgrowth in control mice but not in TRA-8 treated mice (c., upper panel),and H&E staining of tumor (c., lower panel). SCID mice were injectedintravenously with 10⁶ Jurkat cells and treated with a single dose ofTRA-8 on the second day after injection. Seven days later, spleen cellswere harvested, stained with anti-human CD3 antibody and analyzed byflow cytometry (d.), or by immunohistochemistry (e).

[0031]FIG. 7 shows expression of cell surface DR5 in RA (A) and OA (B)synovial cells. 1×10⁶ primary cultured synovial cells were stained withaffinity-purified TRA-8 and followed by PE-conjugated goat anti-mouseIgG1 antibody. 10,000 viable cells analyzed by FACSvantage.

[0032]FIG. 8 is a series of graphs showing cell viability as a functionof TRAIL and TRA-8 concentration induced apoptosis of representativestrains of RA (A) and OA (B) synovial cells with various concentrationsof the recombinant soluble TRAIL (the open circles) or affinity-purifiedTRA-8 (the closed circles). Cell viability is the percentage of the cpmof treated cells versus the cpm of untreated cells.

[0033]FIG. 9 is a series of graphs showing the caspase dependence ofDR5-mediated apoptosis of RA synovial cells. RA synovial cells (RA512)are incubated with 50 ng/ml of soluble Fas ligand (open squares),anti-Fas antibody (CH-11) (closed squares), soluble TRAIL (opencircles), or anti-DR5 antibody (TRA-8) (closed circles) in the presenceof variable concentrations of caspase inhibitors. After overnightculture, cell viability is determined by ATPLite.

[0034]FIG. 10A is an electrophoretic gel-shift assay indicating NFkbactivation. RA1016 cells are incubated with 20 ng/ml TNF-a, 50 ng/mlsoluble TRAIL or 50 ng/ml TRA-8 for indicated time points before beingsubjected to electrophoresis. FIGS. 10B and C are graphs showing theproduction of MMP-1 and MMP-3.1×10⁶/ml of indicated RA synovial cellsare incubated with the indicated concentrations of TNF-a (the opencircles), TRAIL (the open triangles) or TRA-8 (the closed circle). Afterovernight culture, the culture supernatants are collected. The levels ofMMPs in culture supernatants are determined by ELISA.

[0035]FIG. 11. TRA-8 does not induce hepatocellular toxicity. (a.)Normal liver tissues do not express DR5. The paraffin sections of twonormal liver tissues, one hepatocellular carcinoma tissue, and thecytospin preparation of HepG2 cells were prepared for H&E staining, andcorresponding frozen sections were stained with TRA-8. (b.) Flowcytometry analysis of cell surface expression of DR5. Hepatocytes,isolated from two normal liver tissues and from a case of hepatocellularcarcinoma tissue, and HepG2 cells were stained with TRA-8, anti-Fasantibody (DX2) or an isotype control antibody. The solid histogramsindicate TRA-8 or DX2 staining, and the open histograms are thecorresponding isotype controls.

[0036]FIG. 12. TRAIL but not TRA-8 induces hepatocellular toxicity.Fresh normal human hepatocytes were maintained in Hepatocyte CultureMedium. (a.) Apoptosis of hepatocytes was induced with 1 μg/ml solubleTRAIL plus crosslinker or TRA-8 for the indicated time points. Cellviability was determined by ATPLite. The results are presented aspercent viable cells compared to the medium control. The shaded barsindicate TRAIL and the black bars indicate TRA-8. (b.) The condensednuclei of hepatocytes were stained with Hoechst 33352 and analyzed byflow cytometry. (c.) Effect of cycloheximide on hepatocytes apoptosis.Hepatocytes were cultured in control medium or with 1 μg/ml TRAIL orTRA-8 in the presence (closed bars) or absence (open bars) of 1 μg/mlcycloheximide for 8 hours. Cell viability was determined by ATPLite. Theresults are presented as mean±SEM of triplicate cultures of twoexperiments. d. A comparison of the susceptibility of normal hepatocytesto DR5 and Fas-mediated apoptosis. Freshly isolated hepatocytes wereincubated with indicated concentrations of soluble TRAIL, TRA-8, solubleFasL or the anti-Fas mAb CH11 for 6 hours. Cell viability was determinedby ATPLite assay. The results are presented as the percentage of viablecells compared to medium control. For normal hepatocytes, the mean±SEMof four normal individuals is presented. The results of hepatocellularcarcinoma cells from one patient and HepG2 cells are presented as theaverage of triplicate cultures.

[0037]FIG. 13. TRAIL induces hepatitis. B6 mice were intravenouslyinoculated with 10⁹ pfu of adenoviral vector encoding the full length ofhuman TRAIL under the control of the “Tet-on” transcriptional element.TRAIL expression was induced by the indicated dose of tetracycline. (a.)Northern blot analysis of human TRAIL expression in the liver. 24 hoursafter inoculation of vector and induction with tetracycline, total RNAwas isolated from the livers and probed with human TRAIL cDNA orβ-actin. (b.) Serum levels of AST. 24 hours after transduction of TRAIL,serum levels of AST were determined. (c.) TRAIL-mediated cell death ofadenoviral vector infected hepatocytes: B6 mice were intravenouslyinoculated with tetracycline-inducible adenoviral vector. 48 hours afterinoculation, hepatocytes from inoculated and non-inoculated control micewere isolated and incubated with indicated concentrations of TRAIL for 8hours (left panel). Cell viability of hepatocytes was determined by theATPLite assay. Mice, inoculated with adenoviral vector as above, wereintravenously injected with 10 μg of soluble human TRAIL 48 hours later.Serum levels of AST were measured at 24 hours after TRAIL injection(right panel). (d. and e.) Histology analysis of liver damage induced byTRAIL. The livers were collected at 24 hours (d.) or 7 days (e.) aftertransduction with TRAIL. The paraffin sections were H&E stained, andphotographed at 100× (top panel) and 400× (lower panel).

[0038]FIG. 14 is a series of graphs showing that activated T cells and Bcells purified from human PBMC express increased levels of DR5 asdetermined by flow cytometry for resting (unfilled) and activated(shaded) cells.

[0039]FIG. 15 is viability graphs as a function of TRA-8 concentrationfor the purified T cells and B cells depicted in FIG. 14 that have beenstimulated for 48 hours with anti-CD3 or anti-μ, with activated andblast cells collected by different density of Ficoll-Paque. Viability isdetermined by ATPLite assay.

[0040]FIG. 16 is a histogram and flow cytometry plots showing CD3expression in a gated lymphocyte population for NK cell depletedNOD/SCID mice injected with human PBMC and TRA-8 or IgG (control).

[0041]FIG. 17 shows CD3 and TUNEL stained cellular micrographs for mousespleen tissue as detailed in Example 13.

[0042]FIG. 18 shows cycotoxicity plots for chronic lympholytic leukemia(CCL) and normal B cell humans in the presence of TRA-8, BISVIII, andthe combination thereof.

[0043]FIG. 19(a) shows specific binding of 2E12 to DR4. ELISA plateswere coated with the soluble form of human TRAIL receptor-human IgG1 Fcfusion proteins as indicated and incubated with indicated concentrationof mAb 2E12, followed by HRP-conjugated anti-mouse IgG1. The reactionwas developed with TMB substrate buffer and OD values were measured at450/650 nM.

[0044]FIG. 19(b) shows 2E12 binds cell surface DR4. Cos-7 cells weretransfected with the vector containing the full length cDNA for DR4(solid histogram) or control vector (open histogram). The transfectedcells were stained with 10 μg/ml 2E12 and PE-conjugated anti-mouse IgG1.Cells were analyzed by flow cytometry.

[0045]FIG. 19(c) shows apoptosis-inducing activity of 2E12. Human RamosB lymphoma cells were incubated overnight with the indicatedconcentrations of 2E12 in the presence of 2 μg/ml of anti-mouse IgG1.Cell viability was determined by the ATPLite assay. (d) Caspaseactivation induced by 2E12. Ramos cells were treated with 2E12 andanti-mouse IgG antibody for the indicated time points. Caspaseactivation and PARP cleavage were determined by Western blot analysisusing specific anti-caspase or PARP antibodies.

[0046]FIG. 20 shows the effect of 2E12 and adriamycin in athymic nudemice bearing breast cancer xenografts. 2LMP cells (3×10⁷) were injectedsubcutaneously into athymic nude mice on day 0. Two groups of mice wereinjected intraperitoneally with 200 μg 2E12 on days 7, 10, 14, 17, 21,and 24. Two groups of mice received i.v. adriamycin (6 mg/kg) on days 8,12, and 16. One group of mice received no antibody. Data are expressedas the average change in tumor size relative to size on day 7 (n=8mice/group).

[0047]FIG. 21 shows the effect of TRA-8, 2E12 and adriamycin in athymicnude mice bearing breast cancer xenografts. 2LMP cells (3×10⁷) wereinjected s.c. into athymic nude mice on day 0. Two groups of mice wereinjected i.p. with 200 μg TRA-8 and 2E12 on days 7, 10, 14, 17, 21, and24. Two groups of mice received i.v. adriamycin (6 mg/kg) on days 8, 12,and 16. One group of mice received no antibody. Data are expressed asthe average change in tumor size relative to size on day 7 (n=8mice/group).

[0048]FIG. 22A shows flow cytometry analysis of DR5 cell surfaceexpression in a panel of human breast cancer cell lines. Breast cancercells were harvested using EDTA and stained with 10 μg/ml TRA-8 mAb for1 h at 4° C. followed by PE-conjugated goat anti-mouse IgG1, thenanalyzed using FACScan and CellQuest software. Thick histograms indicateTRA-8 staining and thin histograms indicate incubation with mouse IgG1isotype control antibody. FIG. 22B shows cytotoxicity of TRA-8 to humanbreast cancer cell lines. Cells were trypsinized and replated at adensity of 1,000 cells/well in a 96-well plate. TRA-8 antibody was addedafter plating cells, and incubated for 24 h at 37° C. Cell viability wasassessed 24 h after TRA-8 addition using the ATPLite assay. ATP levelsare reported relative to untreated control cells as the mean and SE from2-3 independent experiments, each done in triplicate.

[0049]FIG. 23A shows cytotoxicity of TRA-8 and adriamycin combinationtreatment of human breast cancer cell lines. Cells (1,000/well) wereexposed to various concentrations of adriamycin for 24 h at 37° C.beginning 24 h after plating cells. TRA-8 was added 24 h afteradriamycin addition, and ATP levels were determined 24 h later. Valuesrepresent the mean and SE of triplicate determinations from 2-4independent experiments each done in triplicate, and are reportedrelative to untreated control cells. FIG. 23B shows cytotoxicity ofTRA-8 and paclitaxel combination treatment of human breast cancer celllines. Cells (1,000/well) were exposed to various concentrations ofpaclitaxel for 24 h at 37° C. beginning 24 h after plating cells. TRA-8was added 24 h after paclitaxel addition, and ATP levels were determined24 h later. Values represent the mean and SE of triplicatedeterminations from 2-4 independent experiments each done in triplicate,and are reported relative to untreated control cells.

[0050]FIG. 24 shows the effect of TRA-8 on tumor growth in athymic nudemice bearing established 2LMP human breast cancer xenografts. 2LMP cells(3×10⁷) were injected s.c. on day 0. Two groups of mice were injectedi.p. with 200 μg or 600 μg TRA-8 on days 7, 10, 14, 17, 21, and 24. Onegroup of mice received no antibody. The data represent the averagechange in tumor size (product of two diameters) relative to size on day7 (n=8 mice/group).

[0051]FIG. 25 shows the effect of TRA-8 and adriamycin on tumor growthin athymic nude mice bearing breast cancer xenografts. 2LMP cells(3×10⁷) were injected s.c. into athymic nude mice on day 0. Two groupsof mice were injected i.p. with 200 μg TRA-8 on days 7, 10, 14, 17, 21,and 24. Two groups of mice received i.v. adriamycin (6 mg/kg) on days 8,12, and 16. One group of mice received no antibody. Data are expressedas the average change in tumor size (product of two diameters) relativeto size on day 7 (n=6-8 mice/group).

[0052]FIG. 26 shows the effect of TRA-8 and paclitaxel in athymic nudemice bearing breast cancer xenografts. 2LMP cells (3×10⁷) were injecteds.c. into athymic nude mice on day 0. Two groups of mice were injectedi.p. with 200 μg TRA-8 on days 7, 10, 14, 17, 21, and 24. Two groups ofmice received i.v. paclitaxel (20 mg/kg) on days 8, 12, 16, 20, and 24.One group of mice received no antibody. Data are expressed as theaverage change in tumor size (product of two diameters) relative to sizeon day 7 (n=8 mice/group).

[0053]FIG. 27 shows the effect of TRA-8, adriamycin, and ⁶⁰Co radiationon tumor growth in athymic nude mice bearing breast cancer xenografts.2LMP cells (3×10⁷) were injected s.c. into athymic nude mice on day 0.Three groups of mice were injected i.p. with 200 μg TRA-8 on days 7, 10,14, 17, 21, and 24. Two groups of mice received i.v. adriamycin (6mg/kg) on days 8, 12, and 16. Four groups of mice received 3 Gy ⁶⁰Coradiation on days 9 and 17. One group of mice received no antibody. Dataare expressed as the average change in tumor size (product of twodiameters) relative to size on day 7 (n=8 mice/group).

DETAILED DESCRIPTION OF THE INVENTION

[0054] The failure to delete cells is due to defects in the apoptosisinducing system which are associated with defects illustrativelyincluding expression or function of the ligand, the receptor, or theintracellular regulatory and effector molecules. The present inventionaffords a method to correct a deficient apoptosis inducing system aswell as to elucidate the specific defects inherent in a given defectiveapoptosis inducing system.

[0055] The present invention relates to a new class of monoclonalantibodies that have selective in vivo and in vitro apoptosis inducingactivity against specific TRAIL receptors, including DR5, DR4, DcR1 andDcR2. Thus, the antibodies of the present invention specifically bindone of the TRAIL receptors. By “selectively binding” or “specificallyrecognizing” means that the antibody binds only one TRAIL receptor andshows little or no binding to other types of TRAIL receptors usingtraditional Western blot analysis. A DR5 antibody of the presentinvention binds DR5 selectively and shows no binding above about 1.5times background for DR4, DcR1 or DcR2. Similarly, a DR4 antibody of thepresent invention binds DR4 selectively and shows no binding above about1.5 times background for DR5, DcR1 or DcR2. The present invention hasutility as a reagent for apoptosis signaling research, as well asutility as a therapeutic effective against cells expressing TRAILreceptors, illustratively including broad classes of cancer cells, cellsshowing disregulation of the apoptosis system, activated lymphocytes orother activated immune cells (e.g., lymphoid cells and myeloid cells),virally infected cells, and abnormally proliferating synovial cells(e.g., rheumatoid arthritis synovial cells, including inflammatorysynovial cells, activated lymphoid and myeloid cells in the synovium,macrophage-like synoviocytes, and fibroblast-like synoviocytes) ofautoimmune diseases. Antibodies according to the present invention arespecific in binding particular types of TRAIL receptors in spite of thehomology there between. The inventive antibodies afford targetedapoptosis of only those cells expressing a target TRAIL receptor oralternatively, blocking TRAIL apoptosis of cells expressing a targetreceptor.

[0056] A DR5 monoclonal antibody or a DR4 monoclonal antibody of thepresent invention serves as a potent inducer of apoptosis in cellsexpressing DR5 or DR4, respectively, in vitro and as a potent inducer ofapoptosis in vivo. Humanized fragmentary CDR sequences engrafted onhumanized antibody backbones and fusion protein DR5 or DR4 antibodies ofthe present invention exhibit similar apoptotic properties.

[0057] To date, no monoclonal antibody is available which binds to cellsurface DR5 and which, even at low concentrations, induces apoptosis ofcells expressing DR5 both in vitro and in vivo in the absence of acrosslinker. The present invention includes a DR5 antibody operative asa therapeutic agent in the treatment of a variety of diseases. Althoughsoluble TRAIL has been shown to be effective in induction of apoptosisof tumor cells in vivo, the killing activity appeared to be very lowwith the large and repeated doses often being required (13). The presentinvention provides a purified antibody which binds a TRAIL receptor DR5,wherein said antibody, in its soluble form at low concentrations, has invivo and in vitro apoptosis-inducing activity in target cells expressingDR5. In a preferred embodiment, the purified antibody binds the TRAILreceptor DR5 in the absence of antibody crosslinking. Preferably, theantibody does not induce significant apoptosis of normal fibroblastcells. Preferably, the apoptosis-inducing activity is characterized byless than 60%, 50%, 40%, 30%, 20%, 10%, 5% viability, or any percentagein between, of the target cells at antibody concentrations of less thanabout 0.1, 1, 5, 10, or 20 μg/ml or any concentration in between. Thepurified antibody specifically binds TRAIL receptor DR5 and does notbind TRAIL receptors DR4, DcR1, or DcR2 upon routine Western blotanalysis. In a preferred embodiment, the antibody is a monoclonalantibody, preferably having the same epitope specificity as mouse-mousehybridoma TRA-8 having ATCC Accession Number PTA-1428.

[0058] TRA-8, one of a series of DR5 antibodies according to the presentinvention, is pharmaceutically effective in animals carrying a human DR5transgene and also has utility in establishing a model for theinvestigation of the role of DR5 and TRAIL.

[0059] Various embodiments of the invention provide antibodies thatinduce apoptosis in the presence or absence of crosslinking. Forexample, a preferred embodiment of DR5 antibody (e.g., TRA-8) inducesapoptosis in the absence of crosslinking. Other embodiments provideantibodies that induce apoptosis in the presence of crosslinkers,including, for example, a preferred embodiment of the DR4 antibody(2E12).

[0060] Thus, the invention provides a purified antibody whichspecifically binds a TRAIL receptor DR4, wherein said antibody, in itssoluble form, has in vivo and in vitro apoptosis-inducing activity intarget cells expressing DR4. As one embodiment, the antibody is amonoclonal antibody having the same epitope specificity as hybridoma2E12 having ATCC Accession Number PTA-3798, deposited on Oct. 24, 2001,having designated name “2E12 Hybridoma Clone Against Human DR4,” onbehalf of The UAB Research Foundation. 2E12, one of a series of DR4antibodies of the present invention, is pharmaceutically active inreducing tumor size, as compared to untreated control animals orcompared to the tumor size before treatment, in vivo in animals with DR4expressing cancers.

[0061] Both antibodies to DR4 and DR5 are effective in soluble form atlow doses, by low doses is meant at doses or concentrations of less thanabout 0.01 to about 1 μg/ml in vitro and less than about 1-10 mg/kg invivo. A preferred feature of the antibodies of the present invention isthat they induce apoptosis selectively to cells expressing DR5 or DR4receptors, without inducing apoptosis in normal, non-activated,non-transformed hepatocytes, fibrocytes, synoviocytes, etc. An antibodyaccording to the present invention raised against a TRAIL receptor isharvested according to the present invention from an experimental animalbut can be made by any methods of antibody production or synthesis knownin the art. By humanizing the antibody according to the presentinvention to maintain receptor binding activity while eliciting adiminished and therapeutically tolerable immune response within a humansubject, a humanized anti-TRAIL receptor antibody according to thepresent invention is used as therapeutic agonist or antagonist for agiven TRAIL receptor. The present invention being operative as an invivo therapeutic since secondary crosslinking of the anti-TRAIL receptorantibody, optionally, is not required.

[0062] The present invention extends beyond a single anti-TRAIL receptorantibody having agonist or antagonistic apoptotic effects. Rather, twoor more anti-TRAIL receptor antibodies are brought into contact with acell culture in vitro or a subject body tissue in vivo to create anenhanced treatment. By “enhanced treatment” is meant any additive,synergistic, or potentiating effect. For example, glioma cell line U87and hematopoietic cell lines U937 and Molt-4 are responsive to exposureto a synergistic exposure to agonistic anti-DR4 and anti-DR5 antibodieswhereas exposure to agonistic anti-DR5 antibody alone shows only limitedsuccess in inducing apoptosis.

[0063] Additionally, antagonistic anti-TRAIL receptor antibodies haveparticular utility in the present invention when an antibody is specificto binding one of the decoy receptors DcR1, DcR2 or OPG. Selectiveblocking of a decoy receptor with an antibody according to the presentinvention has the effect in cell types expressing decoy receptors ofshifting the TRAIL binding equilibrium towards those TRAIL receptorscapable of transducing the apoptosis signal. Thus, in another combinedtherapy according to the present invention, a decoy receptor bindingantibody sensitizes an expressing cell towards agonistic apoptosissignal transducing TRAIL receptor binding.

[0064] In another embodiment, the present invention affords a method ofelucidating agonistic and antagonistic epitopes of a given TRAILreceptor. Further, polymorphisms between individuals associated with agiven TRAIL receptor are elucidated according to the present inventionthrough the use of a panel of monoclonal antibodies each having adiffering variable or CDR region. A characterized panel of monoclonalantibodies provides the ability to define agonistic and antagonisticepitopes and polymorphisms. Thus, a panel of monoclonal antibodiesaccording to the present invention has utility in drug discovery and/orsubject screening for disease proclivity.

[0065] Still another embodiment of the present invention involves fusionproteins including an antigenic fragment of a TRAIL receptor coupled toan immunoglobulin protein, polypeptide or fragment thereof. A TRAILreceptor fragment being defined as containing a sufficient number ofbases to elicit an immunogenic response to a native TRAIL receptorexpressed on a subject cell surface. A TRAIL receptor fusion fragmentincluding at least ten amino acids. An immunoglobulin fusion protein orfragment thereof is defined herein to include a native or syntheticprotein or polypeptide segment having a sufficient number of amino acidbases to activate an immunogenic cascade response within a subject. Animmunogen of the present invention including a fusion of a TRAILreceptor fragment coupled to an immunoglobin fragment has utility as anin vivo therapeutic to elicit an anti-TRAIL receptor antibody in situwithin a subject.

[0066] In still a further embodiment, the present invention is operativeas a gene therapy. The invention thus provides a method of selectivelyinducing apoptosis in target cells comprising the steps of transfectingthe target cells with a vector comprising an expressible TRAIL receptornucleic acid sequence; expressing on said cells a TRAIL receptor encodedby said TRAIL receptor nucleic acid sequence; and contacting said cellswith an apoptosis-inducing antibody selective for binding said TRAILreceptor. In a gene therapy aspect of the present invention, targetedcells are transfected with a vector carrying an expressible sequencecorresponding to a TRAIL receptor, the vector being conventional andchosen on the basis of the targeted cell susceptibility to the vector.Gene therapy vectors illustratively include adenovirus, pAdCMV5. Uponthe targeted cells or tissue expressing the transfected TRAIL receptor,the cells or tissue are exposed to an antibody according to the presentinvention specific for binding the transfected TRAIL receptor. It isappreciated that the anti-TRAIL receptor antibody is either agonistic orantagonistic thereto consistent with the desired therapeutic result.

[0067] The antibodies of the present invention are also operative inconjunction with a sensitizer. A sensitizer as used herein is defined toinclude any stimulus that induces apoptosis, including ultravioletlight, organic molecules specifically including the class ofbisindolmaleimides, heavy metals and free radical species.

[0068] In the context of cancer therapy, TRA-8, is able to induceapoptosis of most TRAIL-sensitive tumor cells in a caspase-dependentfashion in the absence of the secondary crosslinking. Both TRA-8 and2E12, alone or in combination, exhibit a strong tumoricidal activity invivo. The ability of TRA-8 or 2E12 to induce apoptosis of mostTRAIL-sensitive cells confirms that either DR5 or DR4 alone issufficient to trigger apoptosis. The majority of tumor cells detailedherein express cell surface DR5 and their susceptibility to TRA-8induced cell death paralleled their susceptibility to TRAIL, indicatingthat DR5 is a primary death receptor for TRAIL-mediated apoptosis inmost tumor cells. Similar results were obtained with antibodies specificfor DR4 (e.g., 2E12). Thus, differential expression of DR5 or DR4 bynormal and cancer cells is operative in the selectivity ofTRAIL-mediated apoptosis. TRA-8 bypasses the decoy receptors to induceTRAIL-mediated apoptosis. Only a minority of TRAIL resistant tumor cellsare sensitive to TRA-8, however, indicating that the decoy receptors donot appear to play a major role in the resistance of tumor cells toTRAIL-mediated apoptosis.

[0069] Although previous studies have indicated that systemicadministration of the soluble form of TRAIL in animals does induce tumorregression without causing toxicity, the membrane-bound form of humanTRAIL induces liver damage in mice as shown herein. However, the hepatictoxicity of TRAIL is much less potent than that of Fas ligand asdemonstrated by the lesser susceptibility of normal hepatocytes toTRAIL-induced injury compared to Fas ligand and by the lack of lethalityof TRAIL in vivo. Thus, titration of TRAIL has utility in cancertherapy.

[0070] As detailed herein, the absence of significant levels of DR5protein expression by normal hepatocytes is shown and is associated withhepatocyte resistance to TRA-8 induced apoptosis. Crosslinking of DR5with monoclonal antibody is insufficient to organize the homopolymericforms of the death receptor able to trigger apoptosis. Experiments inmarmoset indicate no evidence of hepatic toxicity of TRA-8administration. Thus, an agonistic monoclonal DR5 antibody is likely tobe more selective and safer than soluble TRAIL as a therapeutic agent.Similarly, DR4 is expressed by transformed or activated cells and is notexpressed in appreciable amounts or only at much lower amounts by normalcells, e.g., fibroblasts. DR4 of the present invention thereof inducesapoptosis of certain target cells without appreciable cell death innon-target cells, like fibroblasts, etc. As used herein the absence ofan effect or the lack of an appreciable or significant effect refers toand includes the complete absence of the effect or an effect that isless than or equal to background or control levels and does not exceedbackground and control levels by more than 1.5 times the background orcontrol level.

[0071] As a screening assay or imaging tool, the present invention iswell suited for detecting small clusters of DR4 or DR5 cells which maystill exhibit normal cell morphology. For example, in situ cell sectionstaining of human cancer cells including lung, prostate and livercancers with labeled antibodies according to the present inventionreadily identifies cancerous cells. The antibodies of the presentinvention are also useful in screening for other disease manifestations,including, for example, various inflammatory and autoimmune diseases,like rheumatoid arthritis. Such screening may be useful even before theonset of other clinical symptoms and could be used to screening subjectsat risk for disease, so that prophylactic treatment can be startedbefore the manifestation of other signs or symptoms. Specifically,cancer cells are observed to express very high levels of DR5 as comparedto normal cells of the same type. Thus, the present invention hasutility as a sensitive screening method for early stage malignancieswithin tissue including at least lung, prostate, colon, blood, cervix,breast, and liver. A therapeutic process is detailed herein for theinhibition of abnormal cell proliferation associated with diseasesillustratively malignant cancers and lymphatic leukemias among others.

[0072] The present invention is detailed herein with particularity to ananti-human DR5 monoclonal antibody designated as TRA-8, having ATCCAccession Number PTA-1428. It is appreciated that the techniques andresults detailed with regard to the agonistic anti-human DR5 monoclonalantibody TRA-8 are wholly extendable and applicable to antagonistic DR5antibodies, as well as antibodies raised against DR4, DCR1 and DcR2acting in both agonistic and antagonistic manners. Thus, the presentinvention is detailed herein with respect to an apoptosis-inducingantibody specific for human DR4. In one embodiment, the antibody has thesame epitope specificity as hybridoma 2E12, which was deposited on Oct.24, 2001, to procure an accession number on behalf of The UAB ResearchFoundation, with the American Type Culture Collection, Rockville, Md.The description of the deposited material was “2E12 Hybridoma CloneAgainst Human DR4,” with the strain designation 2E12 and the referencedocket number as PCT/US01/14151. The levels of expression of anapoptosis receptor, such as Fas, do not necessarily correlate with thesusceptibility of the cells to apoptosis. For TRAIL-mediated apoptosis,it has been suggested that the expression of the decoy receptors forTRAIL influences the susceptibility of the cells. Moreover, it has beensuggested that DR5 must be associated with DR4 for effectivetransduction of the apoptosis signal through FADD and the caspase 8pathway. The availability of agonistic monoclonal anti-DR5 antibodyallowed evaluation of the regulation of DR5 signaling and its relativerole in TRAIL-mediated apoptosis. Comparison of the susceptibility ofthe cells to TRA-8-mediated apoptosis with their susceptibility toTRAIL-mediated apoptosis offers insight into the role of DR5 inTRAIL-mediated apoptosis and the mechanisms that may affectsusceptibility. Similar advantages are provided by the DR4 antibody.

[0073] This advantage generally extends to humanized DR5 and DR4antibodies of the present invention. A molecular clone of an antibody toDR-5, for example, is prepared by known techniques as detailed withrespect to the following Examples. Recombinant DNA methodology (33) isoperative herein to construct nucleic acid sequences which encode amonoclonal antibody molecule or antigen binding region thereof.

[0074] The present invention allows the construction of humanized TRAILreceptor antibodies that are unlikely to induce a human anti-mouseantibody (hereinafter referred to as “HAMA”) response (34), while stillhaving an effective antibody effector function. Fully human antibodiescan also be made by immunizing mice capable of making a fully humanantibody (e.g., mice genetically modified to produce human antibodies),screening clones that bind DR5 or DR4, induce apoptosis, and compete forTRA-8 or 2E12 epitope. See, e.g., Lonberg and Huszar (1995) Humanantibodies from transgenic mice, Int. Rev. Immunol. 13:65-93, which isincorporated herein by reference in its entirety for methods ofproducing fully human antibodies. As used herein, the terms “human” and“humanized,” in relation to antibodies, relate to any antibody which isexpected to elicit a therapeutically tolerable weak immunogenic responsein a human subject.

[0075] The present invention provides for a DR5 antibody, a humanizedanti-DR5 antibody, TRA-8 heavy and light chain immunoglobulins andhumanized heavy and light chain immunoglobulins. The invention alsoprovides a DR4 antibody, a humanized DR4 antibody, heavy and light chainimmunoglobulins of the DR4 antibody and humanized heavy and light chainimmunoglobulins, nucleic acids that encode the antibodies and heavy andlight chains, vectors comprising those nucleic acids, and cellscomprising the vectors. Certain truncations of these proteins or genesperform the regulatory or enzymatic functions of the full sequenceprotein or gene. For example, the nucleic acid sequences coding thereforcan be altered by substitutions, additions, deletions or multimericexpression that provide for functionally equivalent proteins or genes.Due to the degeneracy of nucleic acid coding sequences, other sequenceswhich encode substantially the same amino acid sequences as those of thenaturally occurring proteins may be used in the practice of the presentinvention. These include, but are not limited to, nucleic acid sequencesincluding all or portions of the nucleic acid sequences encoding theabove polypeptides, which are altered by the substitution of differentcodons that encode a functionally equivalent amino acid residue withinthe sequence, thus producing a silent change. It is appreciated that thenucleotide sequence of an immunoglobin according to the presentinvention tolerates sequence homology variations of up to 25% ascalculated by standard methods (“Current Methods in Sequence Comparisonand Analysis,” Macromolecule Sequencing and Synthesis, Selected Methodsand Applications, pp. 127-149, 1998, Alan R. Liss, Inc.) so long as sucha variant forms an operative antibody which recognizes a TRAIL receptorDR5. For example, one or more amino acid residues within a polypeptidesequence can be substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs (i.e., aconservative substitution). For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the present invention are proteins or fragments or derivativesthereof which are differentially modified during or after translation,e.g., by glycosylation, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligands, etc. In addition, the recombinantvector encoding nucleic acid sequences of the antibodies of the presentinvention may be engineered so as to modify processing or expression ofa vector. Other modifications can be made in either the nucleic acid oramino acid sequence without reducing or without substantially reducingapoptosis activity in the antibody. Such modifications can occur in theCDRs or non-CDR regions using techniques routine in the art. See, e.g.,Yang et al. (1995), J. Mol. Biol. 254:392-403, which is herebyincorporated by reference in its entirety for methods of CDR walkingmutagenesis.

[0076] Additionally, an inhibitor encoding nucleic acid sequence can bemutated in vitro or in vivo to create and/or destroy translation,initiation, and/or termination sequences or to create variations incoding regions and/or form new restriction endonuclease sites or destroypre-existing ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to in vitro site directed mutagenesis, J. Biol. Chem.253:6551, use of Tab linkers (Pharmacia), and the like.

[0077] X-ray crystallography data indicate that the antibodyimmunoglobulin fold generally forms a long cylindrical structurecomprising two layers of antiparallel b-sheets, each consisting of threeor four b-chains. In a variable region, three loops from each of the Vdomains of H and L chains cluster together to form an antigen-bindingsite. Each of these loops is termed a complementarity determining region(CDR). The CDRs have the highest variability in amino acid sequence withthe antibody. The portions of the variable region that are not part of aCDR are called “framework regions” (“FR” regions) and generally play arole in maintaining CDR structure. Preferably, all the CDRs from a givenantibody are grafted into an acceptor antibody, in order to preserve thebinding region for the TRAIL receptor epitope region. It is appreciatedthat grafting a portion of the total amount of CDRs into a donor isoperative herein. It is understood that grafting generally entails thereplacement, residue for residue, of one amino acid or region, foranother. However, occasionally, especially with the transfer of aregion, one or more residues may be added or omitted or substitutedtherefor, as desired, and that such deletions and insertions, as well asappropriate replacements and inversions, are within the skill of thosein the art.

[0078] An antibody of the present invention is obtained by, for example,grafting each CDR of L chain and H chain subunit of an anti-TRAILreceptor monoclonal antibody into a corresponding CDR region of a humanantibody, thereby humanizing a mouse monoclonal antibody effectiveagainst a TRAIL-receptor.

[0079] Antibody fragments which contain the idiotype of the molecule arealso generated and operative herein using known techniques. For example,such fragments illustratively include the anti-TRAIL receptor (AB′)2fragment which can be produced by pepsin digestion of the antibodymolecule, the TRAIL receptor antibody AB′ fragments generated throughreduction of the disulfide bridges of the TRAIL receptor (AB′)2fragment, and the antibody fragment which are generated by treating theantibody molecule with papain and a reducing agent.

[0080] The antibodies of the present invention can be made usingnumerous techniques known in the art. By way of example, the anti-DR5monoclonal antibody TRA-8 may be obtained by culturing a hybridomawhich, in turn, may be obtained by immunizing a mouse with human DR5 andsubsequently fusing the spleen cells or lymph node cells from the mousewith mouse myeloma cells.

[0081] Preparation of a monoclonal antibody illustratively involves thefollowing steps:

[0082] a) purification of a biomacromolecule for use as an antigen;

[0083] b) preparation of antibody producing cells, after firstimmunizing an animal using injections of the antigen, bleeding theanimal and assaying the antibody titer, in order to determine when toremove the spleen;

[0084] c) preparation of myeloma cells;

[0085] d) fusing the antibody producing cells and myeloma cells;

[0086] e) selecting a hybridoma producing a desired antibody;

[0087] f) preparing a single cell clone (cloning);

[0088] g) optionally, culturing the hybridoma cells, or growing animalsinto which the hybridoma cells have been transplanted, for large scalepreparation of the monoclonal antibody; and

[0089] h) testing the biological activities and the specificity, orassaying marker agent properties, of the monoclonal antibody thusprepared.

[0090] The procedure for the preparation of a monoclonal antibody isdetailed below with reference to the above described steps. This methodfor preparing an antibody of the present invention is intended only tobe illustrative of the methods of preparation and is not limitedthereto. Other known procedures may be followed, or the following methodmodified, for instance by using antibody producing cells other thanspleen cells and myeloma.

[0091] (a) Preparation of Antigen

[0092] A recombinant protein (hereinafter referred to as “recombinanthuman DR5” or “recombinant human DR4”), effective as the antigen, isobtained by transfecting QBI-293A cells with the expression vectorpAdDR5-IgG for a fusion protein comprising the extracellular domain ofhuman DR5 or DR4 and the Fc region of human IgG1 antibody (hereinafterreferred to as “IgG”), (cf. PTA-1428) to express it by using theADENO-Quest kit (Quantum Biotechnologies Inc., Canada), and collectingand partially purifying the expression product. The plasmid pAdDR5-IgGis constructed by inserting DNA encoding a human DR5 or DR4 and humanIgG fusion protein into pAdCMV5, which is an expression vector foranimal cells. Other materials, such as the DNA encoding DR5 or DR4, thevector, and the host, are operative herein.

[0093] The human DR5 or DR4 and IgG fusion protein produced in theculture supernatant of the QBI-293A cells transfected with the vectorpAdDR5-IgG may be partially purified by ProteinA-Sepharose affinitychromatography or Protein G-Sepharose affinity chromatography, orion-exchange chromatography using a Resource Q column (trade name;Pharmacia).

[0094] Alternatively, purified DR5 or DR4 obtained from the cellmembranes of human cell lines is used as the antigen. Further, since theprimary structures of DR4 and DR5 are known (cf. PTA-1428), a peptidecomprising the amino acid sequence of SEQ ID NO. 1, may be chemicallysynthesized by a known method such as the Sanger method, and used as theantigen.

[0095] (b) Preparation of Antibody Producing Cells

[0096] A mouse is immunized with the immunogen produced in step (a),mixed with an adjuvant, such as Freund's complete or incomplete adjuvantor alum. Other suitable experimental animals illustratively includerats, guinea pigs, rabbits, dogs, chickens, horses, pigs, cows andsheep.

[0097] Suitable administration routes to immunize an experimental animalinclude the subcutaneous, intraperitoneal, intravenous, intradermal, andintramuscular injection routes, with subcutaneous and intraperitonealinjections being preferred.

[0098] Immunizations are optionally performed by a single dose or, byseveral repeated doses at appropriate intervals (preferably 1 to 5weeks). Immunized animals are monitored for antibody titer in theirsera, and an animal with a sufficiently high antibody titer is selectedas the source of antibody producing cells. Selecting an animal with ahigh titer makes the subsequent process more efficient. Cells for thesubsequent fusion are generally harvested from the animal 3 to 5 daysafter the final immunization.

[0099] Methods for assaying antibody titer include various well knowntechniques such as radioimmunoassay (hereinafter, referred to as “RIA”),solid-phase enzyme immunoassay (hereinafter, referred to as “ELISA”),fluorescent antibody assay and passive hemagglutination assay, with RIAand ELISA preferred for reasons of detection sensitivity, rapidity,accuracy and potential for automation.

[0100] Determination of antibody titer may be performed, for example, byELISA, as follows. First, purified or partially purified DR5 or DR4 isadsorbed onto the surface of a solid phase, such as a 96-well ELISAplate, followed by blocking any remaining surface, to which DR5 or DR4has not been bound, with a protein unrelated to the antigen, such asbovine serum albumin (BSA). After washing, the well surfaces arecontacted with serially diluted samples of mouse sera to enable bindingof the DR5 or DR4 antibody in the samples to the antigen. An labeled,anti-mouse antibody, as the secondary antibody, is added to be bound tothe mouse antibody. The label can include an enzymatic label, afluorescent label or other labels known in the art. After washing, theenzyme substrate is added, and antibody titer is estimated bydetermining absorbance change due to color development caused by thealteration of the substrate or the like.

[0101] (c) Preparation of Myeloma Cells

[0102] Cells from established mouse cell lines serve as the source ofmyeloma cells, including for example 8-azaguanine resistant mouse,derived from BALB/c myeloma strains P3X63Ag8U.1 (P3-U1) (35),P3/NSI/1-Ag4-1(NS-1) (36). Sp2/0-Ag14 (SP-2) (37), P3X63Ag8.653 (653)(38) and P3X63Ag8 (X63) (39). The cell line selected is seriallytransferred into an appropriate medium, such as 8-azaguanine medium.8-azaguanine medium includes Iscove's Modified Dulbecco's Medium(hereinafter referred to as “IMDM”) or Dulbecco's, Modified Eagle Medium(hereinafter referred to as “DMEM”). RPMI-1640 medium supplemented withglutamine, 2-mercaptoethanol, gentamicin, fetal calf serum (hereinafterreferred to as “FCS”), and 8-azaguanine. The cells are then transferredto a normal medium, such as ASF104 medium (Ajinomoto, K. K.) containing10% FCS, 3 to 4 days prior to fusion, in order to ensure that at least2×10⁷ cells are available on the day of fusion.

[0103] (d) Cell Fusion

[0104] Lymphocytes and plasma cells obtained from any suitable part ofthe animal are precursor cells to produce the antibody. Lymphocyte orplasma cell sources illustratively include spleen, lymph nodes,peripheral blood, or any appropriate combination thereof, with spleencells being the most common source.

[0105] After the last booster injection, tissue in which antibodyproducing cells are present is removed from a mouse having thepredetermined antibody titer. The currently favored technique for fusionof spleen cells with myeloma cells prepared in step c), employspolyethylene glycol.

[0106] The fusion technique includes washing spleen and myeloma cellswith serum-free medium (such as RPMI 1640) or phosphate buffered saline(hereinafter referred to as “PBS”) so that the number ratio of spleencells to myeloma cells is approximately between 5:1 and 10:1, and thencentrifuged. After the supernatant has been discarded and the pelletedcells sufficiently loosened, 1 ml of serum-free medium containing50%(w/v) polyethylene glycol (m.w. 1,000 to 4,000) is added dropwisewith mixing. Subsequently, 10 ml of serum-free medium is slowly addedand then centrifuged. The supernatant is discarded again, and thepelleted cells are suspended in an appropriate amount of HAT mediumcontaining a solution of hypoxanthine, aminopterin and thymidine(hereinafter referred to as “HAT”) and mouse interleukin-2 (hereinafterreferred to as “IL-2”). The suspension is then dispensed into the wellsof culture plates (also referred herein simply as “plates”) andincubated in the presence of 5% v/v CO₂ at 37° C. for about 2 weeks,with the supplementary addition of HAT medium as appropriate.

[0107] e) Selection of Hybridomas

[0108] When the myeloma strain used is resistant to 8-azaguanine, i.e.,it is deficient in the hypoxanthine guanine phosphoribosyl transferase(HGPRT) enzyme, any unfused myeloma cells and any myeloma-myelomafusions are unable to survive in HAT medium. On the other hand, fusionsof antibody producing cells with each other, as well as hybridomas ofantibody producing cells with myeloma cells can survive, the former onlyhaving a limited life. Accordingly, continued incubation in HAT mediumresults in selection of only the desired hybridomas.

[0109] The resulting hybridomas grow into colonies that are thentransferred into HAT medium lacking aminopterin (HT medium). Thereafter,aliquots of the culture supernatant are removed to determine anti-Fasantibody titer by, for example, ELISA. When the above-mentioned fusionprotein is used as the ELISA antigen, it is also necessary to eliminateclones producing an antibody which is specifically bound to the Fcregion of human IgG1. The presence or absence of such a clone may beverified, for example, by ELISA using Fas-IgG1 or IgG1, as the antigen.

[0110] (f) Cloning

[0111] Hybridomas which have been shown to produce specific antibodies,using a method similar to that described in step b) to determineantibody titer, are then transferred to another plate for cloning.Suitable cloning methods include: the limiting dilution method, in whichhybridomas are diluted to contain one cell per well of a plate and thencultured; the soft agar method in which colonies are recovered afterculturing in soft agar medium; a method of using a micromanipulator toseparate a single cell for culture; and “sort-a-clone,” in which singlecells are separated by a cell sorter.

[0112] The cloning procedure according to, for example, the limitingdilution method is repeated 2 to 4 times for each well demonstrating anantibody titer, and clones having stable antibody titers are selected asanti-DR5 monoclonal antibody producing hybridomas. Hybridomas producingan anti mouse DR5 antibody are selected by a similar method to obtain ananti-DR5 monoclonal antibody producing cell line.

[0113] The mouse-mouse hybridoma TRA-8 which is a basis for antibodiesof the present invention was deposited with American Type CultureCollection on Mar. 1, 2000, and has the accession number PTA-1428. The2E12 hybridoma was deposited with American Type Culture Collection onOct. 24, 2001, as described above and has the accession number ATCC No.PTA-3798. Accordingly, when preparing an antibody using the mouse-mousehybridoma TRA-8 or any other established hybridoma, the preparation maybe performed by following a procedure starting from the step (g) below,with the steps (a) to (f) omitted.

[0114] (g) Culture of Hybridoma to Prepare Monoclonal Antibody

[0115] The hybridoma obtained by the cloning is then cultured in normalmedium, not in HT medium. Large-scale culture is performed by rollerbottle culture, using large culture bottles, or by spinner culture. Thesupernatant from the large-scale culture is then harvested and purifiedby a suitable method, such as gel filtration, which is well known tothose skilled in the art, to obtain an DR5 or DR4 monoclonal antibodywhich is a basis for antibodies of the present invention. The hybridomamay also be grown intraperitoneally in a syngeneic mouse, such as aBALB/c mouse or a nu/nu mouse, to obtain ascites containing a DR5 or DR4monoclonal antibody in large quantities. Commercially availablemonoclonal antibody purification kits (for example, MAbTrap GII Kit;Pharmacia) are conveniently used to purify the harvested antibodies.

[0116] Monoclonal antibodies prepared as above have a high specificityfor human DR5 or DR4, respectively.

[0117] (h) Assay of Monoclonal Antibody

[0118] Suitable identification methods of the isotype and the subclassof the monoclonal antibody include the Ouchterlony method, ELISA andRIA. Preferably, a commercial kit is used for identification, such as aMouse Typer Kit (trade name; BioRad).

[0119] Quantification of protein may be performed by the Folin-Lowrymethod, or by calculation based on the absorbance at 280 nm (1.4(OD280)=Immunoglobulin 1 mg/ml).

[0120] Identification of the epitope that the monoclonal antibodyrecognizes are performed as follows. First, various partial structuresof the molecule that the monoclonal antibody recognizes are prepared.The partial structures are prepared by the method wherein variouspartial peptides of the molecule are synthetically prepared by knownoligopeptide synthesis technique, or the method wherein DNA encoding thedesired partial polypeptide is incorporated in a suitable expressionplasmid, and is expressed in a suitable host, such as E. coli, toproduce the peptides. Generally, both methods are frequently used incombination for the above object. For example, a series of polypeptideshaving appropriately reduced lengths, working from the C- or N-terminusof the antigen protein, can be prepared by established geneticengineering techniques. By establishing which fragments react with theantibody, an approximate idea of the epitope site is obtained.

[0121] The epitope is more closely identified by synthesizing a varietyof smaller oligopeptides corresponding thereto or mutants of the peptideusing established oligopeptide synthesis techniques to determine abinding property of the peptides to the anti-DR5 monoclonal antibody,for example, which is a basis for preparation of the antibody of thepresent invention and a competitive inhibition of binding of the peptideto an antigen with the monoclonal antibody. Commercially available kits,such as the SPOTs Kit (Genosys Biotechnologies, Inc.) and a series ofmultipin peptide synthesis kits based on the multipin synthesis method(Chiron Corp.) may be conveniently used to obtain a large variety ofoligopeptides.

[0122] An antibody of the present invention has the various functionalproperties a) to f) described below, each of which is verified by, forexample, a method described herein below.

[0123] a) Specific Binding of TRA-8 to Cells Expressing Human DR5.

[0124] A unique feature of the present invention is the ability to bindcell surface DR5. This is demonstrated by flow cytometry analysis ofcells expressing DR5. First, specific cell surface binding of DR5 isconfirmed by the COS-7 cells transfected with the full-length cDNAencoding human DR5. Specifically, TRA-8 only recognizes COS-7 cellstransfected with DR5 but not empty control vector or vector encodingDR4. Second, three different origins: hematopoietic, glioma, andprostate cancer of human malignant tumor cells are tested. The majorityof these transformed tumor cells expressed significant levels of cellsurface DR5, although expression levels varied largely. Third, twopanels of human primary synovial fibroblast cells from RA and OApatients are examined. All RA synovial cells expressed significantlyhigher levels of DR5 compared to OA cells.

[0125] b) Induction of Apoptosis of Human Malignant Tumor Cells in vitroin the Absence of Crosslinking.

[0126] The ability of an antibody raised according to the presentinvention to recognize TRAIL receptor and to directly induce apoptosisof malignant human tumor cells is determined by cell viability assay(ATPLite) during in vitro culture of cells with various concentrationsof an antibody, specifically TRA-8. The majority of tumor cells aresusceptible to TRA-8 induced apoptosis. For some cells, TRA-8 exhibiteda strong apoptosis-inducing activity, for example, TRA-8 is able toinduce apoptosis of human Jurkat cells within the pg/ml levels.Importantly, TRA-8 induced apoptosis did not require crosslinking, andin most cells, TRA-8 exhibited a stronger apoptosis-inducing activitythan the recombinant soluble TRAIL in the presence of the enhancer.

[0127] c) Tumoricidal Activity of TRA-8 in vivo.

[0128] Tumoricidal activity of TRA-8 is evaluated in two SCID/humantumor cell models. First, SCID mice are intravenously inoculated withhuman leukemia Jurkat cells, and treated with a single dose (100 μg) ofTRA-8. The results show that the majority of implanted Jurkat cells areeliminated from the peripheral blood and spleen by the treatment withTRA-8, as determined by flow cytometry analysis and in situimmunohistochemical straining of Jurkat cells. Second, human astrocytomacells, 1321N1, are subcutaneously inoculated in SCID mice, and thetumor-bearing mice are treated with a single dose of TRA-8. The growthof implanted 1321N1 cells is significantly inhibited in TRA-8 treatedmice as determined by the sizes of tumor and histological analysis.

[0129] d) Identification of RA Synovial cells by TRA-8

[0130] The primary synovial cells isolated from 8 RA and 4 OA patientsare tested for cell surface expression of DR5. TRA-8 is able topositively strain all RA cells but negatively stain all OA cells. Thus,RA is differentiated from OA by the surface expression of DR5 asdetected by TRA-8.

[0131] e) Induction of Apoptosis in RA Synovial Fibroblast Cells byTRA-8

[0132] The ability of TRA-8 to induce apoptosis of RA synovial cells isdetermined by cell viability assay during in vitro culture in thepresence of various concentrations of TRA-8. All RA cells exhibited highto intermediate levels of susceptibility to 100 ng/ml of TRA-8. Incontrast, all OA cells are essentially resistant to TRA-8 inducedapoptosis. Importantly, TRA-8 exhibited a better apoptosis-inducingactivity to RA synovial cells than soluble TRAIL with the enhancer.Moreover, compared to anti-Fas antibody (CH-11), TRA-8 exhibited abetter selectivity to RA synovial cells.

[0133] f) TRA-8 Does Not Induce Production of MMPs in RA Synovial Cells

[0134] Since TRA-8 is able to induce NF-kb activation in RA synovialcells as TNF-a, the effect of TRA-8 on the production of MMP1 and MMP3of synovial cells is determined. While TNF-a induced a dose-dependentincrease of MMPs, TRA-8 is unable to induce any production of MMPs, andin some concentrations, TRA-8 slightly decreased the production of MMPsin RA synovial cells.

[0135] g) TRA-8 Induces Multiple Caspase Activation.

[0136] Since caspases play a crucial role in induction of apoptosis. Theability of TRA-8 to induce caspase activation is determined in humanJurkat cells. When Jurkat cells are incubated with a low dose (50 ng/ml)of TRA-8, the activation of caspase 8, caspase 9, and caspase 3 isobserved as early as 15 minutes after incubation as demonstrated byWestern blot analysis and caspase cleavage analysis. In term of timing,number and strength of caspase activation, antibodies of the presentinvention including the demonstrative antibody TRA-8 exhibited a muchbetter activity than any other known apoptosis-inducing antibodies, suchas anti-human Fas antibody (CH-11).

[0137] The 2E12 antibody specifically binds DR4 in its soluble form, hasin vivo and in vitro apoptosis-inducing activity in target cellsexpressing DR4 (including for example, cancer cells, rheumatoidarthritis synovial cells, activated immune cells like activatedlymphocytes, and virally infected cells), has tumoricidal activity invivo (preferably, in the absence of toxicity to non-tumor cells).Preferably the DR4 antibody of the invention has apoptosis-inducingactivity characterized by less than about 60%, 50%, 40%, 30%, 20%, or10% target cell viability at antibody concentrations of less than 30μg/ml, 3 μg/ml, 0.3 μg/ml, or 0.03 μg/ml and tumoricidal activitycharacterized by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in tumor size. Thus, an antibody of the present invention is asubstance having a property to selectively induce apoptosis inpathogenic cells as shown in effect (a) and (g). Accordingly, it isuseful as a prophylactic and therapeutic agent for diseases associatedwith inappropriate survival of cells or inappropriate proliferation ofcells, such as those attributable to dysregulation of apoptosis systemsincluding the Fas/Fas ligand system.

[0138] The ability of an antibody of the present invention to induceapoptosis is confirmed by culturing cells such as the human leukemiacell line Jurkat (American Type Culture No. TIB-152) and astrocytomacell line 1321NI in medium in which the test sample has been added, anddetermining the survival rate by, for example, an ATPLite assay.

[0139] Antibody of the present invention, especially DR5 and DR4antibodies having almost the same immunogenicity to human as that ofhuman antibodies, is used as an agent for prophylaxis or treatment ofdiseases associated with inappropriate survival or proliferation ofcells, including those attributable to dysregulation of the apoptosissystems in inflammatory and autoimmune diseases illustratively includingsystemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis,graft-versus-host disease, Sjogren's syndrome, pernicious anemia,Addison disease, scleroderma, Goodpasture's syndrome, Crohn's disease,autoimmune hemolytic anemia, sterility, myasthenia gravis, multiplesclerosis, Basedow's disease, thrombopenia purpura, insulin-dependentdiabetes mellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; glomerular nephritis; hypoplastic anemia;rejection after organ transplantation and numerous malignancies of lung,prostate, liver, ovary, colon, cervix, lymphatic and breast tissues. Theantibodies of the present invention can be used to target andselectively induce apoptosis in activated immune cells includingactivated lymphocytes, lymphoid cells, myeloid cells, and rheumatoidsynovial cells (including inflammatory synoviocytes, macrophage-likesynoviocytes, fibroblast-like synoviocytes) and in virally infectedcells (including those infected with HIV, for example) so long as thosetargeted cells express or can be made to express the specific TRAILreceptors (i.e., DR4 or DR5).

[0140] Such a prophylactic or therapeutic agent may be administered invarious forms. Suitable modes of administration include oraladministration, such as by tablets, capsules, granules, powders andsyrups, or parenteral administration, such as by injection orsuppositories.

[0141] The antibody or therapeutic agent may be administered orally,rectally, intracistemally, intraventricular, intracranial, intrathecal,intra-articularly, intravaginally, parenterally (intravenously,intramuscularly, or subcutaneously), locally (powders, ointments, ordrops), by intraperitoneal injection, transdermally, by inhalation or asa buccal or nasal spray. The exact amount of the antibody or therapeuticagent required will vary from subject to subject, depending on the age,weight and general condition of the subject, the severity of the diseasethat is being treated, the location and size of the tumor, theparticular compounds used, the mode of administration, and the like. Anappropriate amount may be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein. Typicalsingle dosages of antibody range from 0.1-10,000 micrograms, preferablybetween 1 and 100 micrograms. Typical antibody concentrations in acarrier range from 0.2 to 2000 nanograms per delivered milliliter. Forinjection into a joint, volumes of antibody and carrier will varydepending upon the joint, but approximately 0.5-10 ml, and preferably1-5 ml, is injected into a human knee and approximately 0.1-5 ml, andpreferably 1-2 ml into the human ankle.

[0142] Depending on the intended mode of administration, the antibody ortherapeutic agent can be in pharmaceutical compositions in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include an effective amount of theselected substrate in combination with a pharmaceutically acceptablecarrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, or diluents. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, which can be administered to an individual along with theselected substrate without causing significant undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.

[0143] Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.

[0144] These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0145] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound is admixed with at least one inert customary excipient(or carrier) such as sodium citrate or dicalcium phosphate or (a)fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

[0146] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weightpolyethyleneglycols, and the like.

[0147] Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

[0148] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil,glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acidesters of sorbitan or mixtures of these substances, and the like.

[0149] Besides such inert diluents, the composition can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0150] Suspensions, in addition to the active compounds, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

[0151] Compositions for rectal administrations are preferablysuppositories which can be prepared by mixing the compounds of thepresent invention with suitable non-irritating excipients or carrierssuch as cocoa butter, polyethyleneglycol or a suppository wax, which aresolid at ordinary temperatures but liquid at body temperature andtherefore, melt in the rectum or vaginal cavity and release the activecomponent.

[0152] Dosage forms for topical administration of a compound of thisinvention include ointments, powders, sprays, and inhalants. The activecomponent is admixed under sterile conditions with a physiologicallyacceptable carrier and any preservatives, buffers, or propellants as maybe required. Ophthalmic formulations, ointments, powders, and solutionsare also contemplated as being within the scope of this invention.

[0153] The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate,acetate, oxalate, valerate, oleate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate,lactobionate, methane sulphonate and laurylsulphonate salts, and thelike. These may include cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, and thelike, as well as non-toxic ammonium, quaternary ammonium and aminecations including, but not limited to ammonium, tetramethylammonium,tetraethylammomium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, S.M. Bargeet al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66:1-19 which isincorporated herein by reference.)

[0154] The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compounds of the above formula,for example, by hydrolysis in blood. A thorough discussion is providedin T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol.14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987.

[0155] A target cell is a cell of an animal illustratively includinghuman, non-human primate, cats, dogs, rat, mouse, guinea pig, rabbit,goat, sheep, cow, horse, chicken, pig, marmoset and ferret.

[0156] In addition, the antibody or therapeutic agent of the presentinvention can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. In general, the solvated forms are considered equivalent to theunsolvated forms for the purposes of the present invention.

[0157] Antibody molecules are purified by known techniquesillustratively including amino absorption or amino affinitychromatography, chromatographic techniques such as high pressure liquidchromatography, or a combination thereof.

[0158] Another aspect of the present invention includes a pharmaceuticalproduct for use in delivering biologically active anti-TRAIL receptorantibody or humanized anti-TRAIL receptor antibody to a vertebrate. Thepharmaceutical product includes a pharmaceutically effective quantity ofanti-TRAIL receptor antibody or fragment thereof, a pharmaceuticallyacceptable carrier, and a container enclosing the carrier and theantibody in a sterile fashion.

[0159] In a preferred embodiment of the invention, a pharmaceuticallyeffective amount of an antibody of the invention inhibits cellproliferation or induces apoptosis by contact with a target cell or withtarget cells. A pharmaceutically effective amount or quantity of anantibody recognizing either DR5 or DR4 or a humanized antibodyrecognizing either DR5 or DR4 is an amount administered to an individualsufficient to cause a desired effect. As used herein, the terms“pharmaceutically effective amount” and “therapeutic quantity” aresynonymous. Desired effects of administration of a pharmaceuticallyeffective amount of DR5 or DR4 recognizing antibodies include death ofone or more target cells, growth inhibition of one or more target cells,stimulation of DR5 or DR4, respectively, binding to DR5 or DR4,respectively, and increased NFkB levels or activity in a target cell. Atarget cell is a cell that expresses DR5 or DR4 and illustrativelyincludes abnormally growing cells and tumor cells such as papillomas andwarts; breast cancer, colon cancer, hepatomas, leukemias, lung cancer,melanoma, myelomas, osteosarcomas, ovarian cancer, pancreatic cancer,prostate cancer, cancer of the head and neck, thyroid cancer, uterinecancer, tumors of the brain such as astrocytomas, activated immune cells(e.g., activated lymphocytes, lymphoid and myeloid cells), inflammatorycells, rheumatoid arthritis synovial cells, and virally infected cells.In vivo, the target cell is a cell of an individual with a pathologicalcondition, including those where cell proliferation is abnormal ordysregulated such as malignant or benign cancer and rheumatoidarthritis.

[0160] In another preferred embodiment, the target cell is alsocontacted by a therapeutic agent.

[0161] A therapeutic agent is a compound or composition effective inameliorating a pathological condition. An illustrative example of atherapeutic agent includes an anti-cancer compound.

[0162] An anti-cancer compound is a compound or composition effective ininhibiting or arresting the growth of an abnormally growing cell. Apharmaceutically effective amount of an anti-cancer compound is anamount administered to an individual sufficient to cause inhibition orarrest of the growth of an abnormally growing cell. Illustrativeexamples of anti-cancer compounds include: bleomycin, carboplatin,chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin,dactinomycin, diethylstilbestrol doxorubicin, etoposide, 5-fluorouracil,floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine,teniposide, 6-thioguanine, vincristine and vinblastine. Further examplesof anti-cancer compounds and therapeutic agents are found in The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J. and Sladek et al. Metabolism and Action of Anti-CancerDrugs, 1987, Powis et al. eds., Taylor and Francis, New York, N.Y.

[0163] Antibody of the present invention can be further combined withother therapies, such as chemotherapy and/or radiotherapy in thetreatment of malignancy, and therapeutic efficacy can be enhanced byapoptosis-inducing compounds, such as bisindolylmaleimide VIII (BisVIII)or other sentizing agents like SN-50 or LY294002. Thus, in oneembodiment, the antibody or antibodies of the present invention can becombined with BisVIII and a chemotherapeutic (e.g., adriamycin). Inanother embodiment, the antibody or antibodies of the present inventioncan be combined with a non-steroidal anti-inflammatory drug (e.g.,sulindac sulfide or other COX-1 or COX-2 inhibitors) a chemotherapeutic(e.g., adriamycin). In the treatment of other diseases, e.g., autoimmuneand inflammatory diseases, combinations of treatment can also be used.For example, an antibody can be administered in conjunction with othertherapeutic agents like chemotherapeutic agents, anti-inflammatoryagents, DMARDs, methotrexate, bisindolylmaleimide VIII or othersensitizing agent, and the like. Radiotherapy can also be combined withother therapeutic agents in the treatment of inflammatory and autoimmunediseases. One skilled in the art would adapt the form of radiotherapy tothe specific inflammatory or autoimmune disease (e.g., radiationsynovectomy in the treatment of arthritis).

[0164] Therapy using an antibody of the present invention can also becombined with therapy using another antibody of the invention. Forexample, an antibody to DR5 can be administered to a subject in needthereof along with, prior to, or following administration of an antibodyto DR4. Such combined antibody therapy can be further combined withadministration of one or more therapeutic agents (e.g.,chemotherapeutics, doxorubicin, and/or methotrexate), radiotherapy, orboth.

[0165] Compared to previously published anti-DR5 antibody (24), theapoptosis-inducing activity of the demonstrative TRA-8 antibody of thepresent invention is very strong, and is able to induce apoptosis ofJurkat cells with the pg/ml levels in vitro and demonstrates superior invivo tumoricidal activity as compared to previously reported solubleTRAIL. The intravenous administration of a single dose of TRA-8 issufficient to inhibit the growth of both solid tumor and hematopoietictumor cells, whereas induction of in vivo tumor regression with thesoluble TRAIL requires much high dose (500 μg every day for 10 days).The anti-TRAIL receptor antibodies of the present invention appear to beas safe as soluble TRAIL since exemplary antibody TRA-8 does not induceapoptosis of non-transformed fibroblast cells. Similar results have beenobserved with DR4 antibodies.

[0166] Vectors of the present invention include a nucleic acid sequenceencoding a heavy or light chain immunoglobulin of a DR5 or DR4 antibodyoperably linked to a regulatory element such as a promoter or enhancer.“Operably linked” refers to an arrangement of nucleotide sequencesconfigured so as to perform their usual function. Thus, a regulatoryelement operably linked to a nucleotide sequence encoding a polypeptideis capable of directing the transcription, replication and/ortranslation of the polypeptide. It will be recognized by those skilledin the art that a single vector optionally includes coding sequences forboth a heavy and a light chain immunoglobulin of a DR5 or DR4 antibody.In one embodiment the vectors encode humanized light or heavy chainimmunoglobulins.

[0167] The following examples are set forth below to illustrate themethods and results according to the present invention. These examplesare not intended to be inclusive of all aspects of the presentinvention, but rather to illustrate representative methods and results.These examples are not intended to exclude equivalents and variations ofthe present invention which are apparent to one skilled in the art.

EXAMPLE 1 Preparation of DR5 Antigen

[0168] 1.1 Cloning of DR5 cDNA

[0169] DNA encoding the human DR5 protein is cloned by the followingRT-PCR method using:

[0170] a) Template

[0171] The total RNA of HeLa cells is extracted by using TRIzol Reagent(GIBCO BRL). The template for the PCR reaction used cDNA that isobtained by using the First-Strand cDNA synthesis kit (AmershamPharmacia Biotech) according to the instruction manual provided with thekit.

[0172] b) PCR Primers

[0173] The following oligonucleotide primers are synthesized for thePCR: (DR5p1: SEQ ID No. 1) 5′-gacgatgcccgatctactttaaggg-3′; (DR5p2: SEQID No. 2) 5′-ccactgggtgatgttggatggg-3′;

[0174] Unless otherwise specified, all oligonucleotides in theseExamples are synthesized by Lifetechnologies. All oligonucleotides arestored at −20° C. after being dissolved in distilled water.

[0175] c) PCR Reaction

[0176] Composition of the PCR reaction solution:

[0177] template cDNA, 5 μl of total 33 μl reaction

[0178] primer DR5p1, 10 pmol;

[0179] primer DR5p2, 10 pmol;

[0180] 10× concentrated PCR buffer (provided with the kit), 10 μl;

[0181] dNTPs (each 2.5 mM), 4 μl; and

[0182] Taq polymerase (Promega), 5 units.

[0183] Sterile distilled water is added to the solution to a totalvolume of 100 μl. Unless otherwise specified, dNTPs are an equimolarmixture of dATP, dCTP, dGTP and dTTP (2.5 mM each).

[0184] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 2 minutes, after which a cycle of heating to 94° C.for 30 sec, 52° C. for 1 minute and 72° C. for 3 minutes, is repeated 40times. After completion of this procedure, the reaction solution isheated at 72° C. for 10 minutes.

[0185] The amplified DNA fragments, thus obtained, are separated on a 1%agarose gel containing 0.25 ug/ml ethidium bromide. The bands determinedto contain the desired DNA fragments are cut out using a razor blade andthe DNA is recovered therefrom using the Gene Clean kit (BIO101). TheDNA fragment is cloned using the TA Cloning Kit (Invitrogen, Calif.).This is performed as follows.

[0186] The DNA fragment recovered from the PCR reaction solution,together with 50 ng of pCR2.1 vector which is provided with the TACloning kit, is mixed with 1 μl of 10×ligase reaction buffer (6 mMTris-HCl (pH 7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mMβ-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine, and 0.1 mg/mlbovine serum albumin), to which 4 units of T4 DNA ligase (1 μl) has beenadded. The total volume of the mixture is adjusted to 10 μl with steriledeionized water, and the resulting ligase solution is incubated at 14°C. for 15 hours. After this time, 2 μl of the ligase reaction solutionis added to 50 μl of competent E. coli strain TOP10F′, which is providedwith the TA cloning kit and brought to competence in accordance with theinstruction manual, to which 2 μl of 0.5 M β-mercaptoethanol has beenadded, and the resulting mixture is kept on ice for 30 minutes, then at42° C. for 30 seconds, and again on ice for 5 minutes. Next, 500 μl ofmedium containing 2% v/v tryptone, 0.5% w/v yeast extract, 0.05% w/vsodium chloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, and20 mM glucose (hereinafter referred to as “SOC” medium) is added to theculture, and the mixture is incubated for 1 hour at 37° C. with shaking.After this time, the culture is spread on an L-broth agar plate (1% v/vtryptone, 0.5% w/v yeast extract, 0.5% w/v sodium chloride, 0.1% w/vglucose, and 0.6% w/v bacto-agar (Difco)), containing 100 μg/ml.Ampicillin resistant colonies appearing on the plate are selected andscraped off with a platinum transfer loop, and cultured in L-brothmedium containing 100 μg/ml ampicillin at 37° C., overnight, withshaking at 200 r.p.m. After incubation, the cells are harvested bycentrifugation, from which plasmid DNA is prepared by the alkali method.EcoRI-EcoRI DR5cDNA fragment from the thus obtained plasmid is subclonedinto pcDNA3 plasmid (Invitrogen, Calif.). The full length of the DR5gene in pcDNA3 are sequenced and matched the published sequence. Thethus obtained plasmid is designated as plasmid pcDNA3-DR5.

[0187] 1.2 Construction of DR5-IgG Expression Vector

[0188] In order to obtain a soluble form of human DR5 lacking thetransmembrane domain, an expression plasmid vector is constructed. Thisvector is designed to encode a fusion protein comprising theextracellular domain of human DR5 fused to the human IgG1 Fc DNA (41).DNA encoding the human DR5 lacking the transmembrane domain is obtainedby the following PCR reaction.

[0189] a) Template

[0190] The template for the PCR reaction used pcDNA3-DR5.

[0191] b) PCR Primers

[0192] The following oligonucleotide primers are synthesized for thePCR: (DR5p1: SEQ ID No. 1) 5′-gacgatgcccgatctactttaaggg-3′; (DR5p3: SEQID No. 3) 5′-ggatccgtggacacattcgatgtc-3′;

[0193] Unless otherwise specified, all oligonucleotides in theseExamples are synthesized by Lifetechnologies. All oligonucleotides arestored at −20° C. after being dissolved in distilled water.

[0194] c) PCR Reaction

[0195] The PCR reaction is conducted and amplified DNA isolated as perExample 1.1(c).

[0196] The thus obtained plasmid is designated as plasmid pCR-ΔDR5. TheBamHI-EcoRI fragment encoded human Fe fragment which is recovered frompmFas-hIgG1Fc is subcloned into BamHI and EcoRI multi-cloning sites ofpcDNA3. The plasmid thus obtained is designated pcDNAFc. Furthermore,the BamHI-BamHI fragment encoding the human soluble DR5 region which isrecovered from pCR-ΔDR5 is subcloned into the BamHI site of pcDNAFcplasmid. The thus obtained plasmid is designated as plasmidpcDNAΔDR5-Fc. The EcoRI fragment encoding the human soluble DR5-humanIgG Fe region which is recovered from the pcDNAΔDR5-Fc plasmid is bluntended by using the DNA polymerase Klenow fragment (GIBCO BRL) and thensubcloned into the shuttle vector pAdCMV5 (Quantum Biotechnologies Inc.,Canada) which is blunt ended after cutting by BamHI. The plasmid thusobtained is designated pAdΔDR5-Fc.

[0197] 1.3 Expression and Purification of the Human DR5-IgG1 FusionProtein

[0198] QBI-293A cells (provided with the ADENO-Quest Kit) areco-transfected with pAdΔDR5-Fc and QBI-viral DNA (provided with theADENO-Quest Kit) using the ADENO-Quest kit (Quantum BiotechnologiesInc., Canada) according to the instruction manual. The recombinant virusplaques are cultured and screened for expression of DR5-IgG fusionprotein by ELISA analysis of the supernatant. The positive plaques areamplified in QBI-293A cells and stored at −80° C. as virus stock. Fiftydishes (150 mm) of QBI-293A cells are transfected with pAdΔDR5-Fcrecombinant virus at 10 m.o.i. (Multiplicity of Infection). The culturemedia are harvested after transfection for 48 hours.

[0199] The transfected cells having the DR5-IgG gene are grown to a celldensity of 1×10⁶ cells/ml by incubation in 500 ml of DMEM (GIBCO)medium, containing 10% v/v FCS, at 37° C. in an atmosphere of 5% v/v CO₂for 2 days. The culture is then centrifuged (1,000 r.p.m., 5 minutes)and the supernatant collected. The purification of DR5-IgG from thesupernatant is achieved using ProteinA-Sepharose CL-4B affinitychromatography (Pharmacia) under the following conditions:

[0200] column: ProteinA-Sepharose CL-4B column (column size 2 ml;Pharmacia);

[0201] elution buffer: 0.1 M glycine (pH 2.4), 0.15 M NaCl;

[0202] neutralization buffer: IM Tris-HCl (pH 8.5).

[0203] After all of the supernatant is applied to the column, it iswashed three times with 20 ml of PBS and then 1 ml of elution buffer isadded 10 times. The optical density of each eluted fraction (1 ml) ismeasured. The second fraction through the fifth fraction (withOD₂₈₀≧0.1) are collected and after addition of 100 μl of neutralizationbuffer, the eluates are placed separately in dialysis tubing, and theeluates dialyzed against 1 liter of PBS (pH 7.5) at 4° C. The dialysisbuffer being changed twice.

[0204] The eluates are then assayed for expression of the DR5-IgG geneproduct by ELISA. First, 100 μl of each fraction are placed separatelyinto wells of a 96-well microplate (Costar) and incubated at 37° C. for1 hour. After this time, the solution in the wells is removed, and theplate is washed 3 times with 100 μl/well of PBS containing 0.1% v/vTween 20 (hereinafter referred to as “PBS-Tween”). After washing, PBScontaining 2% w/v bovine serum albumin (hereinafter referred to as“BSA”) is added in quantities of 100 μl/well, and the plate is thenincubated at 37° C. for 1 hour. After this time, the wells are washed afurther 3 times with 100 μl/well of PBS-Tween, after which 100 μl/wellof a solution of anti-human IgG1 monoclonal antibody diluted 1000-foldwith PBS-Tween is added to each well, and the plate is once againincubated at 37° C. for 1 hour. The wells are then washed 3 times with100 μl/well of PBS-Tween. 3,3′,5,5′-Tetramethyl-benzidine (hereinafterreferred to as “TMB”) liquid substrate system (Sigma) is then added inan amount of 100 μl/well and the plate is allowed to stand at roomtemperature for 5 minutes and then the reaction stopped by adding 100μl/well of 0.2N H₂SO₄. The absorbance of each well is read at 450 nm toestimate the concentration of the bound antibody, using the absorbanceat 650 nm as the control reading. The absorbance is measured using amicroplate reader (Molecular Devices). The production of DR5-IgG1 isconfirmed using this ELISA method. The molecular weight of the expressedDR5-IgG1 fusion protein is determined using western blotting analysis inwhich anti-human IgG1 mAb (Sigma) is used to detect the antibody on thegel. The molecular weight of the expressed DR5-IgG1 fusion protein hasan approximate molecular weight of 50 kDa. The purity achieved beinggreater than 90% as evaluated by analysis on SDS-PAGE and detection ofthe protein by Coomassie blue staining.

EXAMPLE 2 Generation of Monoclonal Antibodies Against Human DR5

[0205] 2.1 Immunization

[0206] Female, Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) of 6-8weeks of age, are immunized with the affinity-purified human DR5/hIgG1fusion protein. For the initial foot-pad immunization, the fusionprotein (50 μg) is emulsified in Freund's complete adjuvant (Difco,Detroit, Mich.). The mice are then boosted with four injections of 50 μgof fusion protein administered without adjuvant every other day. Threedays after the last injection, lymphocytes from the local lymph nodesare fused with NS-1 myeloma cells, and the hybridomas are cultured inF104 media supplemented with 10% fetal calf serum. Positive hybridomasare selected by ELISA in which the plates are coated either with 1 μg/mlDR5/hIgG1 or the same amount of Fas/hIgG1 as a control. The isotype ofthe hybridomas is determined by ELISA using a panel of mouse Igisotype-specific goat antibodies (Southern Biotechnology, Birmingham,Ala.). Monoclonal antibodies are purified by affinity chromatographyusing immobilized anti-mouse IgG1 or protein G (Sigma).

[0207] 2.2 Cell Fusion

[0208] On the third day after the booster injection, the local lymphnodes are removed from the mouse and placed into 10 ml of serum-freeRPMI 1640 medium (GIBCO BRL) containing 50 units/ml penicillin, 50 μg/mlstreptomycin, and 300 μg/ml L-glutamic acid, and disrupted by passingthe organ through a mesh (Cell Strainer; Falcon) using a spatula. Theresulting cell suspension is centrifuged to pellet the local lymph nodescells which are then washed twice with serum-free RPMI medium. Thewashed cells are then resuspended in serum-free RPMI medium and counted.

[0209] In the meantime, myeloma NS 1 cells (American Type CultureCollection TIB-18) had been grown to a cell density not exceeding 1×10⁸cells/ml in ASF104 medium (Ajinomoto, K. K.) containing 10% v/v FCS(Gibco BRL) (“ASF medium with serum”) at 37° C. under 5% v/v CO₂, andthese are likewise disrupted, washed, resuspended and counted.

[0210] An amount of the NS1 cell suspension calculated to contain 3×10⁷cells is mixed with an amount of the spleen cell suspension calculatedto contain 3×10⁸ cells. The resulting mix is centrifuged and thesupernatant discarded. The following steps of the cell fusion areperformed whilst at all times keeping the plastic tube containing thepellet at 37° C. in a beaker of warm water.

[0211] One ml of 50% (w/v) polyethylene glycol 1500 (Boehringer Manheim)is then slowly added to the tube, all the while stirring the pelletusing the tip of a pipette. Subsequently, 1 ml of serum-free RPMImedium, prewarmed to 37° C., is slowly added in 2 portions, followed bythe addition of a further 7 ml of serum-free RPMI medium. The resultingmix is then centrifuged, the supernatant discarded and 10 ml of HATmedium containing 10% v/v FCS are added while stirring gently with thetip of a pipette. A further 20 ml of HAT medium containing 10% v/v FCSis added, and the suspension is dispensed into 96-well cell culturemicroplates at 100 μl/well and incubated at 37° C. in an atmosphere of5% v/v CO₂. After 7 or 8 days, 100 μl/well of fresh HAT medium are usedto replace medium in any wells exhibiting a yellowish hue. The fusioncells from these wells are cloned by limiting dilution as describedbelow.

[0212] 2.3 Cloning by Limiting Dilution

[0213] Thymuses from 4 to 10 week-old female BALB/c mice (from JapanSLC, Inc.) are removed, disrupted on a mesh (Cell Strainer; Falcon) asdescribed above, and the disrupted cells are washed twice with HT mediumcontaining 10% v/v FCS. An amount of thymus cells corresponding to thosefrom one mouse is suspended in 30 ml of HT medium containing 10% v/v FCSto produce a feeder cell suspension. The fusion cell preparationobtained above in Example 2.2 is diluted with this feeder cellsuspension 10- to 100-fold, and further diluted serially with the feedercell suspension to make suspensions having fusion cell densities of 5, 1and 0.5 cells/ml. The thus prepared samples are dispensed into wells of96-well cell culture microplates at 100 μl/well and incubated for 5 daysat 37° C. under 5% v/v CO₂.

[0214] 2.4 Screening

[0215] The culture supernatants from the growing hybridomas are screenedby ELISA using plates coated either with 1 μg/ml DR5/hIgG1 or the sameamount of Fas/hIgG1 (41) as a control. The bound antibodies are detectedusing horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulins(Southern Biotechnology. Birmingham, Ala.) with TMB (Sigma, St Louis,Mich.) as the substrate. Purified DR5-IgG1 at a concentration of 1 μg/mlor the same amount of Fas-hIgG1 are introduced into a well of a 96-wellELISA/RIA STRIP PLATE (Costar, N.Y.). The plate is kept standing at 4°C. overnight to allow adsorption of the protein onto the well surface.After this time, the solution in the wells is discarded and each well iswashed 3 times with PBS-Tween. Then, 100 μl of PBS containing 1% (w/v)bovine serum albumin (A3803; Sigma Chemicals Co.) is added to each welland the plate is incubated at 37° C. for 1 hour. The wells are thenwashed a further 3 times with PBS-Tween, and then 50 μl of each culturesupernatants from the growing hybridomas is added to each well. Theplate is then incubated at 37° C. for 1 hour, and the wells are againwashed 4 times with PBS-Tween. After washing, 50 μl of horseradishperoxidase labeled goat anti-mouse immunoglobulin antibody (SouthernBiotechnology. Birmingham, Ala.), diluted 1000-fold with PBS, is addedper well, and the plate is again incubated at 37° C. for 1 hour, afterwhich the wells are washed 4 times with PBS-Tween.3,3′,5,5′-Tetramethyl-benzidine (TMB) liquid substrate system (Sigma) isthen added in an amount of 100 μl/well and the plate is allowed to standat room temperature for 5 minutes and then the reaction stopped byaddition of 100 μl/well of 0.2N H₂SO₄. The absorbance of each well at450 nm (control 650 nm) is measured using a microplate reader (MolecularDevices) and fusion cells are selected from the sample which had theabsorbance (450 nm-650 nm, OD values; >0.5) clearly higher than those towhich no fusion cells supernatant had been added (OD values; ≈0.03).Furthermore, the culture supernatants from the growing hybridomas arealso functionally screened by measuring the apoptosis-inducing activityusing Jurkat cell. Fifty μl of RPMI medium containing Jurkat cells (1000cells per well) and 5 uM Bisindolylmaleimide VIII (BisVIII, Alexis, SanDiego, Calif.) are added in 96-well plates in the presence of 50 ul ofthe culture supernatants from the growing hybridomas. The cells arecultured in a humidified incubator at 37° C. overnight. Apoptosis isdetermined by cell viability using the ATPLite kit as instructed by themanufacturer (Packard Instruments), and the samples are counted usingthe TopCounter (Packard Instruments).

[0216] 2.5 ELISA Binding of TRAIL and TRA-8 to the Receptors

[0217] ELISA plates are coated with 2 μg/ml of DR4-Ig or DR5-Ig fusionprotein overnight. After blocking with 3% BSA, the soluble TRAIL-FLAG orTRA-8 is added at indicated concentrations and incubated at 37° C. forone hour. The binding of TRAIL or TRA-8 is detected by HRP-conjugatedanti-Flag antibody (Alexis) or HRP-conjugated anti-murine IgG1 (SouthernBiotechnology), respectively. The reactions are developed by TMBsubstrate buffer and measured by the Benchmark Microplate Reader(BioRad). The Kd values are estimated by the one-site binding model ofnon-linear regression using GraphPad Prism software (GraphPad Software,San Diego, Calif.). For competitive ELISA, 100 ng/ml TRAIL-FLAG is addedand incubated in the presence of various concentrations of TRA-8. Thebinding of TRAIL is determined as above.

[0218] 2.6 Cloning

[0219] The steps described in Examples 2.3 and 2.4 above are repeated 5times for the cells selected in 2.4, thereby enabling the selection ofseveral hybridoma clones each of which produced a single antibody thatbound DR5-IgG but did not bind Fas-IgG. As a result of this selectionprocedure, a mouse-mouse hybridoma, designated TRA-8 and producing anantibody binding to DR5-IgG, but not Fas-IgG, is obtained. Thishybridoma, TRA-8, was deposited with the American Type CultureCollection on Mar. 1, 2000, and has been assigned accession No.PTA-1428.

[0220] The subclass of the antibody produced by the mouse-mousehybridoma TRA-8 (hereinafter referred to simply as “TRA-8”) isdemonstrated to be IgG1, K, after testing with a monoclonal antibodyisotyping kit (Pierce).

[0221] Using our human DR5-IgG1 fusion protein as immunogen, sevenhybridoma clones are obtained by initial ELISA screening, all of whichare strongly positive for DR5-IgG but not the Fas-IgG fusion protein,indicating that the obtained hybridomas produce antibodies thatrecognize the extracellular portion of DR5 but not the Fc portion ofIgG1 (data not shown).

[0222] 2.7 Western Blot Analysis

[0223] Filters for Western blot analysis of normal human and cancertissue homogenates are purchased from Geno Technology (St Louis, Mo.).Each lane is loaded with an equal amount of protein as determined by ananti-μ-actin antibody. The blots are probed with 1 μg/ml TRA-8overnight, and followed by HRP-conjugated goat anti-mouse IgG1 (SouthernBiotechnology) at room temperature for one hour, and developed bychemiluminescence.

[0224] 2.8 In situ Immunohistochemistry

[0225] Human tissues are obtained from the Tissue Procurement Center ofUAB. Frozen sections are fixed in 70% ethanol, blocked with 10% horseserum in PBS, and then incubated with 10 μg/ml of affinity-purifiedTRA-8 at room temperature for 60 minutes. The anti-mouse IgG ABC kitwith diaminobenzidine (Vector, Burlingame, Calif.) as the colorimetricsubstrate is used to visualize the reactivity.

[0226] 2.9 Analysis of Caspase Activation

[0227] Jurkat cells (1×10⁶/ml) are incubated with 500 ng/ml TRA-8.Aliquots (30 μg of protein) of the cell lysate are separated on 15%SDS-PAGE, blotted onto a nylon membrane, and the blots are probed withanti-caspase 8, 9, and 3 antibodies (BD Pharmingen, San Diego, Calif.)followed by HRP-conjugated secondary antibody and chemiluminescencevisualization of cleaved products. The caspase inhibitor set ispurchased from R&D Systems (Minneapolis, Minn.). Each caspase inhibitoris added into culture at indicated concentrations.

EXAMPLE 3 Purification of TRA-8 Monoclonal Antibody

[0228] The mouse-mouse hybridoma, TRA-8, is grown to a cell density of1×10⁶ cells/ml by incubation in 500 ml of ASF medium, containing 10% v/vFCS, at 37° C. under 5% v/v CO₂ for 5 days. The culture is thencentrifuged (1,000 r.p.m., 5 minutes) and the supernatant collected. Thepurification of TRA-8 from the supernatant is achieved usingProteinG-Sepharose CL-4B affinity chromatography (Pharmacia) under thefollowing conditions:

[0229] column: ProteinG-Sepharose CL-4B column (column size 2 ml;Pharmacia);

[0230] elution buffer: 0.1 M Glycine (pH 2.4), 0.15 M NaCl;

[0231] neutralization buffer: IM Tris-HCl (pH 8.5).

[0232] After all of the supernatant is applied to column, 20 ml of PBSis washed three times and then elution buffer is added in 1 ml volumesfor 10 times. The optical density of each eluted fraction (1 ml) ismeasured. The fractions from No. 2 to No. 5 (>OD₂₈₀=0.1) are collectedseparately.

[0233] After adding 100 μl of neutralization buffer, the eluates areplaced in dialysis tubing separately, and the eluates dialyzed against 1liter of PBS (pH 7.5) at 4° C. The dialysis buffer being changed twice.This sample is assayed for anti-DR5 antibody activity by ELISA using thehuman DR5-IgG fusion protein prepared above using the techniquedescribed above.

EXAMPLE 4 Preparation of DR4 Antigen, DR4-IgG Expression Vector andAnti-DR4 Monoclonal Antibody

[0234] The procedures of Examples 1-3 are repeated with DR4 templatecDNA and primers in place of those detailed in Example 1 to obtain a DR4antigen which is utilized as per Examples 1.2-3 to obtain a monoclonalantibody specific against DR4.

EXAMPLE 5 Monoclonal Antibodies Against DcR1 and DcR2

[0235] Monoclonal antibodies are raised against decoy receptors DcR1 andDcR2 by substituting the corresponding cDNA and primers to create therespective antigens as per Example 1. Expression vectors for DcR1 orDcR2-fusions with immune globulin G and resulting purified monoclonalantibodies are created as per Examples 2 and 3.

EXAMPLE 6 The Specificity of a Monoclonal Antibody

[0236] As all of the receptors for TRAIL and other proteins of the TNFRfamily share significant homology, the specificity of exemplary antibodyTRA-8 for DR5 is determined by western blot analysis using two differenthuman DR5-IgG fusion proteins and soluble, recombinant forms of otherrelated proteins. A first DR5-Ig fusion protein is constructed by fusingcDNA from residues 1-180 of the extracellular portion of DR5 and cDNAencoding the constant region of human IgG1. The fused cDNA is clonedinto a recombinant adenoviral vector (Quantum Biotechnogies, Inc.,Montreal, Canada). The expressed DR5/hIgG1 fusion protein, which had arelative molecular weight of 50 kDa, is purified using an anti-human IgGaffinity column (Sigma, St Louis, Mo.). For western blot analysis ofspecificity, a second recombinant human DR5/IgG1 fusion protein (aa.52-212), as well as TRAIL-R1, R3 and R4 fusion proteins, are purchasedfrom Alexis. The soluble forms of human Fas and TNFR1 are kindlyprovided by Dr. Carl Edwards of Amgen, Inc., Thousands Oaks, Calif.,USA. The soluble recombinant human DR4, DcR1, DcR2, TNFR1, R4, and Fasmolecules used are human IgG1 fusion proteins. 0.5 μg of each protein isseparated by 10% SDS-PAGE and blotted onto a nitrocellulose membrane.The blots are blocked with 5% dry milk in PBS at room temperature forone hour, and probed with 1 μg/ml of purified monoclonal anti-DR5antibody (clone: TRA-8) or 0.1 μg/ml of HRP-conjugated goat anti-humanIgG at 4° C. overnight. Horseradish-peroxidase (HRP)-conjugated goatanti-mouse IgG is used as secondary antibody to detect bound TRA-8. Theblots are developed by chemiluminescence.

[0237] Cos-7 cells transfected with the pcDNA3 vector (Clontech, PaloAlto, Calif.) containing the full-length DR5 or DR4 or empty vector areused for flow cytometry analysis. The full-length cDNA encoding humanTRAIL or murine Fas ligand is cloned into the pTRE vector down-stream ofthe tetracycline-controllable promoter (Clontech). The XhoI-HindIIIfragments of pTRE-hTRAIL or pTRE-mFasL are further cloned into theadenoviral shuttle vector pAdBN (Quantum Biotechnologies, Inc.). The 293host cells are co-transfected with the linearized pAd-TRE-hTRAIL orpAd-TRE-mFasL and the large fragment DNA of adenovirus. The expressionof functional human TRAIL or murine Fas ligand from the recombinantvirus plaques is screened using a ⁵¹Cr-release assay with Jurkat as thetargets.

[0238] TRA-8 reacted strongly with the DR5-IgG fusion protein (˜50 kDa),which is used for immunization as shown in FIG. 1a, DR5, #1, and weaklywith the second DR5-IgG fusion protein (˜60 kD) as shown in FIG. 1a,DR5, #2. There is no significant binding of TRA-8 to DR4, DcR1, DcR2,Fas (CD95) or TNFRI. These results indicate that TRA-8 recognizes theepitopes that are specific for DR5 but not shared by the other membersof the family.

[0239] TRA-8 does not react with other members of the TNF receptorsuperfamily, such as Fas (CD95) and TNF receptor I, nor does TRA-8cross-react with the murine homologue of DR5 as shown by opticalabsorbance ratios for 450 nm and 650 nm, wherein lower panel numbers 1-7(FIG. 1a, the column 8 of lower panel). Soluble TRAIL and TRA-8 boundcomparably to immobilized DR5 (FIG. 1b, left panel). In contrast, TRAILbound to DR4, but TRA-8 did not exhibit any binding activity to DR4(FIG. 1b, middle panel). The Kd values for the binding of TRAIL andTRA-8 to DR5 are estimated at 59 nM and 3 nM, respectively. Importantly,TRA-8 efficiently competing with TRAIL for binding to DR5 but not forbinding to DR4, as shown in competitive ELISA (FIG. 1b, right panel).These results establish the specificity of TRA-8 for human DR5.

[0240] TRA-8 is able to detect cell surface expression of DR5, with flowcytometric analysis indicating specific binding to the cell surface ofCos-7 cells transfected with full-length human DR5, but not of Cos-7cells transfected with DR4 or empty vector (FIG. 1c). Similarly, in situimmunohistochemistry with TRA-8 demonstrated reactivity with Cos-7 cellstransfected with full-length DR5 DNA but not with those transfected withcontrol vector (FIG. 1d). TRA-8 does not induce apoptosis ofuntransfected Cos-7 cells, and RT-PCR of RNA from Cos-7 cells usingpaired primers encoding human DR5 showed that no specific PCR products.Further functional analysis using human Jurkat cells as targets showedthat, in the absence of crosslinking, TRA-8 strongly induces cell death,demonstrated by three different assays for cell viability includingATPLite, MTT and PI exclusion (FIG. 1e). Greater than 50% of Jurkatcells are killed by nanogram levels of TRA-8 as shown by ATPLite assay.The killing activity of TRA-8 is specific for DR5 as it could be blockedby DR5-Ig but not DR4-Ig fusion protein (data not shown). Cleavage ofcaspases 8, 9, and 3 could be detected by western blot analysis as earlyas 30 minutes after TRA-8 treatment of Jurkat cells (FIG. 1f), and celldeath of Jurkat cells is completely inhibited by the general caspaseinhibitor (Z-VAD) (FIG. 1g). Individual caspase inhibitors for caspase8, 3, 9, and 10 partially inhibited cell death, further indicating thatTRA-8-mediated cell death is primarily through a caspase-dependentapoptotic mechanism.

EXAMPLE 7 Flow Cytometric Analysis of the Expression of Cell SurfaceDR5: A Major Death Receptor on Many Tumor Cells But Not on Normal Cells

[0241] The ability of TRA-8 to bind DR5 expressed on the cell surfaceand the specificity of this reaction is then assessed using COS-7(American Type Culture Collection No. CRL-1651) cells transfected withthe expression vector containing the full-length human DR5 or DR4 cDNAor empty vector as control. Phycoerythrin (PE)-conjugated anti-mouseIgG1 (Pharmingen) is used as the second antibody to detect the boundTRA-8. The fluorescence of 1×10⁴ cells is measured, using a flowcytometer (FACSVantage) under the following conditions:

[0242] Excitation wave length: 488 nm;

[0243] Detection wave length; 600 nm.

[0244] Flow cytometry analysis showed that TRA-8 stained approximately30% of COS-7 cells transfected with the DR5 vector as shown in the solidhistogram of FIG. 1c. This percentage parallels the transfectionefficiency as determined by analysis of transfection using greenfluorescent protein (GFP) (data not shown). TRA-8 did not significantlystain cells transfected with either DR4 (the open histogram) or controlvector (the dotted histogram), indicating that TRA-8 is specific forcell surface DR5.

[0245] Although DR5 expression in tumor cells has been studiedextensively at the mRNA level (REFERENCE please insert), the surfaceexpression of DR5 has not been documented. Thus, the availability ofmonoclonal anti-DR5 antibody allows us to examine the surface levels ofDR5, and to correlate the expression with the susceptibility of thecells to TRAIL-mediated apoptosis. The following panel of cells (1×10⁶)is incubated with 10 μg/ml of affinity purified TRA-8 at roomtemperature for 30 min, and then stained with PE-conjugated anti-mouseIgG1 (Pharmingen) for another 30 min. 10,000 viable cells are analyzedusing the FACS vantage flow cytometer under the following conditions:

[0246] Excitation wave length: 488 nm;

[0247] Detection wave length: 600 nm.

[0248] The five hematopoietic cell lines tested are Jurkat, CEM-6,Molt-4, H-9 and U937 cells. DR5 expression is detectable on the surfaceof Jurkat, CEM-6, H-9, and U937 cells but is almost undetectable onMolt-4 cells as shown in FIGS. 2a and 2 a′. Although high levels of DR5RNA expression has been described previously (43), the FACs analysisindicated that these cells do not express high levels of the surfaceDR5. These results indicate that cell surface expression of DR5 does notcorrelate with the transcriptional expression of DR5, which is notunexpected for such a receptor. The level of cell surface expression ofDR5 may be cell lineage-specific since most of the cells ofhematopoietic origin expressed low levels whereas most glioma andprostate cells expressed high levels of DR5.

[0249] TRA-8 monoclonal antibody is used determine the role of DR5 ininduction of TRAIL-mediated apoptosis by examining its cell surfaceexpression among a panel of different types of human tumor cells as wellas the susceptibility of these cells to both TRAIL and TRA-8-mediatedapoptosis. Primary peripheral blood T cells did not express significantlevels of cell surface DR5 and are resistant to both TRAIL andTRA-8-mediated apoptosis (FIGS. 2a, 2 a′ and 3a). Although all five ofthe human T-leukemia cell lines tested expressed detectable albeitrelatively low levels of cell surface DR5, two of them (Jurkat andCEM-6) are highly susceptible to both TRAIL-mediated and TRA-8-mediatedapoptosis, indicating that DR5 alone is sufficient to induce apoptosisof these cells. Molt-4 and U937 cells are partially susceptible toTRAIL-mediated apoptosis but are relatively resistant to TRA-8-mediatedapoptosis, suggesting that other TRAIL receptors might be involved intransduction of an apoptosis signal. H-9 cells are resistant to bothTRAIL and TRA-8-mediated apoptosis, implicating a block mediated by anintracellular anti-apoptosis pathway.

[0250] The panel of cells included the human malignant glioma celllines, Hs683, U251MG, D37MG, D54MG, U373MG, CH235MG, U87 and normalhuman astrocytes, which were provided by Dr. Yancey Gillespie of theNeurosurgery Department of the University of Alabama at Birmingham. Thehuman prostate cancer cell lines, Dul54, PC3 and LnCap, were provided byDr. William Grizzle of the Pathology Department of the University ofAlabama at Birmingham who had obtained the cell lines from the AmericanType Culture Collection. The human leukemia T cell lines, B-celllymphoma, HepG2 Jurkat (American Type Culture Collection TIB-152) andCCRF-CEM CEM-6 (American Type Culture Collection CCL-119); monocyte celllines, U937 (American Type Culture Collection CRL-2367); were purchasedfrom the American Type Culture Collection. All above cell lines arecultured in RPMI 1640 supplemented with 10% FCS. The human astrocytomacell line, 1321N1, was kindly provided by Dr. Richard Jope of PsychiatryDepartment of the University of Alabama at Birmingham, and cultured inDMEM supplemented with 5% FCS.

[0251] Soluble recombinant human TRAIL, purchased from AlexisCorporation (San Diego, Calif.), is a fusion protein comprised of theextracellular domain of human TRAIL (aa residues 95 to 281) fused atN-terminus to a FLAF-tag and an 8 amino acid linker peptide. Unlikepreviously reported His-tagged TRAIL, this preparation of TRAIL alonedoes not induce a strong apoptotic response in Jurkat cells and requiresan anti-FLAG antibody as a crosslinker to enhance apoptosis. Theanti-FLAG antibody was also purchased from Alexis.

[0252] All of the 10 human malignant glioma cells tested expresseddetectable levels of DR5 at the cell surface. Most expressedintermediate to high levels of DR5 as shown in FIG. 2b. Three lines,D-54MG, U373MG and CH-235MG expressed high levels of DR5 while sixlines, Hs-683, U251-MG, D37-MG, U87, SMK1 and 1321N1, expressedintermediate levels of DR5. Only one cell line, H-465 expressed lowlevels of DR5. All three prostate cancer cell lines expressed highlevels of DR5 as shown in FIG. 2c.

[0253] Like the normal primary T cells, primary B cells did not expresssignificant levels of DR5 and did not undergo apoptosis after treatmentwith either TRAIL or TRA-8 (FIG. 2d). Three (SKW6.4, EB-3, and Raji) outof the four B lymphoma cell lines tested expressed relatively highlevels of DR5 and are very susceptible to both TRAIL and TRA-8-mediatedapoptosis. The fourth cell line, Daudi, expressed very low levels of DR5and is much less susceptible to either TRAIL or TRA-8-mediatedapoptosis. Although primary astrocytes did not express detectable levelsof cell surface DR5 (FIG. 2b′), all four glioma cell lines testedexpressed high levels of DR5. The higher level of expression of DR5 onglioma cells than on T and B cells is not accompanied by a significantlygreater susceptibility to TRAIL and DR5-mediated apoptosis, suggestingthat the level of cell surface expression of DR5 is not necessarilycorrelated with the level of apoptosis of tumor cells. RT-PCR, performedto determine message levels of DR4, DR5 and DCR2, detected message inall cells tested (Table 1). However, in general, primary normal cellsexpressed relatively low levels of DR5 compared to transformed tumorcells. TABLE 1 RT-PCR analysis of TRAIL receptor expression* Cells DR5DR4 DcR2 Primary T cells <0.001 <0.001 0.015 Jurkat 0.10 <0.001 0.21CEM-6 0.50 0.59 0.25 Molt-4 0.10 <0.001 0.05 H-9 0.73 0.61 0.07 PrimaryB cells <0.001 <0.001 0.024 SKW6.4 0.95 0.66 0.45 EB3 0.40 <0.001 0.35Raji 0.55 0.11 0.45 Daudi 0.73 0.36 0.63 Normal Astrocytes 0.05 <0.0010.12 SH683 0.56 0.96 0.14 U87 0.44 0.56 0.21 D54 1.15 0.46 0.12 1321N10.25 0.35 0.05

EXAMPLE 8 Induction of Apoptosis in vitro in Malignant Cells

[0254] To determine whether TRA-8 induces apoptosis in transformed cellsin vitro, all DR5-positive tumor cells are examined for theirsusceptibility to apoptosis induced either by TRA-8 or TRAIL.

[0255] Target cells (1×10³ per well) are cultured in 96-well plates inthe presence of the indicated concentrations of soluble TRAIL pluscrosslinker (Alexis) or TRA-8 at 37° C. overnight. Cell viability isdetermined using (1) the ATPLite kit according to the manufacturer'sinstructions (Packard Instruments, Meriden, Conn.); (2) the MTT Cellproliferation/viability kit (Sigma); or (3) PI staining of dead cellsand analyzed by flow cytometry. At end of culture, cells are stainedwith 10 μg/ml PI and PI negative cells are gated as viable cells. Foranalysis of condensed nuclei of hepatocytes, cells are stained with 10ng/ml Hoechst 33352 (Molecular Probes) and analyzed by flow cytometry.

[0256] The TRA-8 antibody is capable of inducing apoptosis in themajority of the malignant human glioma cell lines (9/10), in 2 of the 3prostate cancer cell lines, and in 2 of the 4 DR5-positive hematopoieticcell lines. It did not induce apoptosis in the Molt-4 cell line, whichexpressed almost undetectable cell surface levels of DR5. The levels ofsusceptibility of the cells to TRA-8-mediated apoptosis variedconsiderably among the cell lines, however.

[0257] The variability of the susceptibility of the cells to TRA-8antibody induced apoptosis suggests that although a minimal level ofcell surface expression of DR5 is required, the level of cell surfaceexpression of DR5 is not necessarily the primary determinant ofsusceptibility and other factors influence this process. Although all ofthe glioma cells generally expressed significantly higher levels of thesurface DR5 than did the hematopoietic cells, glioma cell susceptibilityto apoptosis induced by TRA-8 is not proportionally increased comparedto the hematopoietic cells. The susceptibility of five of the gliomacell lines, D-37MG, D54-MG, U373-MG, CH235-MG and 1321N1 toTRA-8-induced apoptosis is high and is equivalent to theirsusceptibility to TRAIL-mediated apoptosis as shown in FIG. 3b. Two ofthe glioma cell lines, H-456 and SMK1, are much less susceptible toapoptosis induced by TRA-8. In the case of the H-456 cells, the surfaceexpression of DR5 is low; however, the surface expression of DR5 on SMK1is similar to the more susceptible cell lines, suggesting that othermechanisms might play a role in the determining the susceptibility toTRAIL-mediated apoptosis. Although all three prostate cancer cell linesexpressed high levels of DR5, the Du145 cells are most sensitive toTRA-8-induced apoptosis, the PC3 cells are partially sensitive whileLnCAP cells are completely resistant as shown in FIG. 3c. Among thehematopoietic cells, it is found that Jurkat and CEM-6 are verysusceptible to TRA-8-apoptosis as shown in FIG. 2a although both thesecell lines had been found to express low levels of DR5. Although DR5 isdetectable on U937 cells, these cells are resistant to TRA-8-inducedapoptosis. Similarly, although the H-9 cells expressed detectable levelsof DR5, H-9 cells are resistant to apoptosis induced by TRA-8. Theseresults implicated the existence of regulatory mechanisms that influenceDR5-mediated apoptosis.

[0258] Additional surface binding anti-DR5 antibodies are produced asper the procedures of Examples 1-3. Two additional anti-DR5 antibodiesdesignated TRA-1 and TRA-10 are studied along with TRA-8 to determinecomparative ability induce apoptosis and thereby act as an agonist orconversely block TRAIL-mediated apoptosis, thereby acting as anantagonist. Human Jurkat cells are used as a target to determine theagonist and/or antagonist activity of the three anti-DR5 antibodiesdenoted TRA-1, TRA-8 and TRA-10. As shown in FIG. 4, cell viability isabout 90%, 70% and 20% for TRA-10, TRA-1 and TRA-8, respectively uponovernight incubation with 2.5 μg per ml. TRA-8 induced a strongapoptotic response in a dose dependent fashion while TRA-1 induced onlya moderate apoptotic response and TRA-10 only induced a weak response.TRA-8 is therefore classified as an agonist anti-DR5 antibody. In FIG.4, the viability of human Jurkat cells is shown as a dose dependentfunction of TRAIL-induced apoptosis. TRA-10 blocked apoptosis of humanJurkat cells to a significant extent in a low dose TRAIL-inducedapoptosis study. Thus, TRA-10 is classified as an antagonist anti-DR5antibody.

[0259] The susceptibility of five of the glioma cell lines, D-37MG,D54-MG, U373-MG, CH235-MG and 1321N1 to TRA-8-induced apoptosis isequivalent to their susceptibility to TRAIL-mediated apoptosis as shownin FIG. 3b, indicating that TRAIL-induced apoptosis in these cells ismediated primarily through DR5. Moreover, two of the glioma cell lines,Hs683 and U251-MG, are resistant to TRAIL-induced apoptosis butpartially sensitive to TRA-8-induced apoptosis, indicating that thedecoy receptors function in these cells and that use of the TRA-8antibody bypassed this regulatory mechanism. In the prostate cancer celllines, despite the varying sensitivity to apoptosis induced by TRA-8,this paralleled the sensitivity of the cells to apoptosis induced byTRAIL, again suggesting that DR5 plays a major role of TRAIL-mediatedapoptosis in the prostate cancer cells. Among the hematopoietic cells,it is found that Jurkat and CEM-6 are very susceptible to both TRA-8 andTRAIL-mediated apoptosis. The level of apoptosis induced by TRA-8 iscomparable to that induced by TRAIL as shown in FIGS. 2a and 3 a′. Onlyone of the glioma cell lines, U87, and two hematopoietic cell lines,U937 and Molt-4, exhibited sensitivity to TRAIL-induced apoptosis butare less sensitive or resistant to TRA-8-induced apoptosis. One cellline, the H-9 cell line, expressed detectable levels of DR5 but areresistant to apoptosis induced by either TRA-8 or TRAIL. While minimallevels of expression of DR5 are required for TRA-8-induced apoptosis,the level of expression of DR5 does not necessarily predict thesusceptibility of the cells to TRA-8 mediated apoptosis; decoy receptorsplay a role in modulating TRAIL-mediated apoptosis in some cells, butdoes not appear to play a major role in most of the cells tested todate; as anticipated the TRA-8 antibody bypasses the effects of thedecoy receptors; functional mutations of the DR5 receptor may occur intransformed cells; and, finally, intracellular regulatory mechanisms maybe as important, or more important than the decoy receptors in definingthe susceptibility of the cells to TRAIL and DR5-mediated apoptosis.

[0260] Previous studies have shown that the mRNA for DR5 is distributedwidely in normal tissues⁷. To evaluate the expression of DR5 at theprotein level, a panel of normal human tissue homogenates (GenoTechnology, St. Louis, Mo.) is probed with the TRA-8 antibody in westernblot analysis. Among nine normal human tissues, brain tissue is weaklypositive (FIG. 5a, lane 2). DR5 protein is not detectable by TRA-8reactivity in liver (lane 1), lung (lane 3), kidney (lane 4), spleen(lane 5), testes (lane 6), ovary (lane 7), heart (lane 8), or pancreas(lane 9). In contrast, all thirteen human cancer tissues stainedpositively with TRA-8 (FIG. 5b), including cancers of the ovary (lane1), lung (lane 2), liver (lane 3), rectum (lane 4), cervix (lane 5),skin (lane 6), testes (lane 7), thyroid (lane 8), uterus (lane 10),stomach (lane 11), laryngopharynx (lane 12), and pancreas (lane 13).Moreover, in situ immunohistochemistry of normal and cancer tissues withTRA-8 confirmed that aside from a few scattered positive cells inspleen, DR5 expression in normal breast, lung and spleen tissues is notdetectable (FIG. 5c). The corresponding cancer tissues including breastinfiltrating ductal carcinoma, small cell lung cancer, and lymphomareacted positively with TRA-8 (FIG. 5d). Among a total of 22 cancertissues examined, 5 of 6 breast cancers, 2 of 2 cancers of the cervix, 4of 5 liver cancers, 5 of 8 lymphomas, 2 of 2 lung cancers, and 2 of 2prostate cancers reacted positively with TRA-8. These results areconsistent with those of the flow cytometry analysis and indicate thatcancerous tissues express higher levels of DR5 protein than do normaltissues.

EXAMPLE 9 Tumoricidal Activity of TRA-8 in vivo

[0261] For various reasons, many agents that show promise in in vitrostudies do not show efficacy in vivo. It is therefore important to testthe efficacy of TRA-8 in an in vivo animal model. To accomplish this theTRA-8 anti-human DR5 antibody is administered to mice bearing humanxenografts that express the human DR5 molecule. The mice used are 6 to 8week-old NOD/SCID mice (Jackson Laboratory), which are inoculatedsubcutaneously with human astrocytoma 1321N1 cells (1×10⁷), orinoculated intravenously with human leukemia Jurkat cells (1×10⁶). Atday 2 after tumor inoculation, mice are inoculated intravenously withTRA-8 (100 μg). Five days after the treatment with TRA-8, 1321N1 tumorgrowth is determined by the size and weight of the tumor mass. Thegrowth of Jurkat cells is determined by the weight of the spleen and thepercentage of human CD3-positive Jurkat cells in the spleen ofinoculated animals. Biopsies of tumor tissues are taken and examinedhistologically.

[0262] Early treatment with a single intravenous dose of 100 μg of TRA-8at one day after tumor inoculation completely inhibited the 1321N1 cellsfrom forming a solid tumor of (FIG. 6a). Late treatment with three dosesof 100 μg TRA-8 at one week after tumor inoculation reduced tumor weight4-fold or more (FIG. 6b). Tumor formation is not visible in animalstreated with TRA-8 at an early time point (FIG. 6c, upper panel).Histologic analysis revealed dramatically degenerated tumor tissue inanimals treated with TRA-8 (FIG. 6c, lower panel). Similarly, TRA-8treatment inhibited population of the spleen by Jurkat cells asdemonstrated by the scarcity of CD3-positive Jurkat cells in the spleen(FIG. 6d, 6 e). Histological analysis of the implanted tumor showed afew tumor cells scattered in the soft tissue in TRA-8-treated animalswhile controls showed the formation of a solid tumor as shown in FIG.6c. In the Jurkat cell model, the number of Jurkat cells in the spleensof TRA-8 treated animals is less than 2% compared to nearly 10% in thespleen of control animals as demonstrated by flow cytometry analysis asshown in FIG. 6a and in situ CD3 staining of FIG. 6c.

[0263] These results confirm the recent demonstration that systemicadministration of cross-linked recombinant TRAIL inhibits growth oftumor in vivo (13). These results indicate that a single dose of TRA-8is highly effective in the elimination of tumor cells in vivo.

[0264] As an anti-human antibody is used in a murine model, the toxicityof the TRA-8 treatment could not be assessed. However, the study ofadministration of TRAIL in vivo indicated that no significant toxicityis associated with this treatment (13).

EXAMPLE 10 RA Synovial Cells are Susceptible to TRAIL and TRA-8-InducedApoptosis

[0265] Most of the prior art studies of TRAIL-mediated apoptosis havefocused on malignant cells. TRAIL-mediated apoptosis according to thepresent invention is also therapeutic in autoimmune and inflammatoryconditions, such as RA.

[0266] 10.1 Flow Cytometric Analysis of the Expression of Cell SurfaceDR5 in RA Synovial Cells

[0267] The expression of DR5 on a panel of eight primary culturedsynovial cells from patients with RA is compared with that on eightprimary cultured synovial cells from patients with osteoarthritis(hereinafter referred to as “OA”). The eight human primary RA synovialcell cultures RA-1014, RA-1016, RA-1021, RA-512, RA-707, RA-811, RA-716,and RA-929 are kindly provided by Dr. M. Ohtsuki (Sankyo Co. Ltd.,Tokyo, Japan) and cultured in DMEM supplemented with 10% FCS,penicillin, streptomycin, and glutamine. The seven OA synovial cellprimary cell cultures are isolated from the synovial tissues of OApatients by a standard collagenase method and cultured under the sameconditions. The passage number of all primary cells is under 10. Theexpression of DR5 is determined by FACs analysis as described in Example5.

[0268] All of the primary cultures of RA cells expressed high levels ofsurface DR5, and there is little variation in the expression levelsamong these synovial cells isolated from different patients as shown inFIG. 7a. In contrast, the expression of surface DR5 on the surface ofsynovial cells isolated from the OA patients is very low or undetectableas per FIG. 7b. SV40-transformed synovial cell are found to express highlevels of DR5 comparable with those exhibited by the RA cells. Incontrast, non-transformed fibroblast cells expressed low levels of DR5comparable to those exhibited by the OA cells in FIG. 7b.

[0269] 10.2 Susceptibility of RA Synovial Cells to Apoptosis Mediated byTRA-8 or TRAIL

[0270] In general, all synovial cells isolated from the RA patients aresusceptible to both TRAIL and anti-DR5 antibody induced apoptosis, andall OA cells are resistant to TRAIL and anti-DR5 antibody inducedapoptosis as per FIG. 8a, b. These studies indicate that the TRA-8antibody targets altered cells in preference to normal cells. Moreover,the pattern of the susceptibility or resistance to apoptosis induced byTRAIL is correlated with that induced by anti-DR5 antibody, indicatingthat the synovial cells primarily utilize DR5 to trigger TRAILapoptosis.

[0271] As described for the malignant cells, the susceptibility toapoptosis induced by TRAIL or anti-DR5 antibody varied among the RAsynovial cells although expressing similar levels of DR5. RA-512 andRA-707 are the most susceptible as over 80% cells are killed byconcentrations of TRAIL or TRA-8 below 20 ng/ml. RA-1014, RA-811,RA-716, and RA929 are among those with the intermediate susceptibilityto TRAIL or TRA-8, with nearly 100% cell death occurring in the presenceof high concentrations (>50 ng/ml) of TRAIL or TRA-8. In RA-1016 andRA1021 cells, although the majority (over 60%) of cells are killed by alow dose of TRAIL or TRA-8, a portion of cells survived in the presenceof high concentrations of TRAIL or TRA-8, indicating that asub-population of cells are resistant to TRAIL-mediated apoptosis. Incontrast, all OA cells are much less susceptible to TRAIL and TRA-8induced apoptosis. No greater than 60% cells are killed in the OA52F andOA69F even in the presence of high concentration of TRAIL or TRA-8.OA72M cells are completely resistant to TRAIL or TRA-8 inducedapoptosis. The SV40 transformed synovial cells are also susceptible toTRAIL and TRA-8 induced apoptosis (data not shown). In contrast, thenon-transformed fibroblast cells appeared to be resistant to TRAIL andTRA-8.

[0272] It has been shown previously that DR5 utilizes a FADD/caspase 8dependent pathway to trigger apoptosis (44). To determine thecaspase-dependence of DR5-mediated apoptosis of RA synovial cells, RAcells are cultured with TRAIL or anti-DR5 antibody in the presence ofspecific caspase inhibitors. Among eight caspase inhibitors tested,caspase 6, 8 and 10 inhibitors are able to inhibit apoptosis of RAsynovial cells induced by both TRAIL and DR5 as shown in FIG. 9,indicating that these three caspases are involved in DR5-mediatedapoptosis.

[0273] 10.3 TRA-8 or TRAIL Induce NF-κb Activation in RA Synovial CellsWithout Increased Release of MMPs

[0274] There is considerable evidence to support the concept that thereare close links between the signaling of apoptosis and the signaling ofproliferation (45). It has been established that DR5 is able to activatea NF-kb pathway in addition to apoptosis signaling transduction, andthat NF-Kb activation may be able to transduce an anti-apoptosis signal.Therefore a gel-shift assay is carried out. Cells are stimulated with 50ng/ml of the recombinant soluble TRAIL, Fas ligand in the presence ofthe 1 mg/ml enhancer, or 50 ng/ml of TRA-8 for the indicated time. Thenuclear extracts are prepared and incubated with the double-stained[³²P]-labeled oligo-DNA probe. The results are analyzed using thecyclone phospha-imager (TopCount NXT, Packard Instrument Company, CT).After RA synovial cells are incubated with TNF-α or TRAIL, NF-κb isactivated in a time-dependent fashion. The TRA-8 antibody is able tostrongly activate NF-κb. In contrast, Fas ligand is unable to induceNF-κb activation as per FIG. 10a.

[0275] Thus, although TRAIL and TRA-8 antibody induce a strong apoptosisresponse in RA synovial cells, they also activate NF-κb, and NF-κbactivation has been believed to contribute to the proinflammatory roleof TNF-α in RA. Thus, it is possible that TRAIL, like TNF-α, may serveas a pro-inflammatory cytokine. To determine whether there is a similarbiological consequence of NF-κb activation induced by TRAIL and TNF-α,the production of MMPs is determined by ELISA. Synovial cells arecultured in medium alone or with 50 ng/ml interleukin 1b, 10 ng/mlTNF-a, 50 ng/ml TRAIL, or 50 ng/ml TRA-8 overnight. The levels of theMMP-1 and MMP-3 in the culture supernatants are determined by the ELISAkits.

[0276] When RA synovial cells are incubated with a proinflammatorycytokine, TNF-α or IL-1b, the production of MMP-1, 3, and 13 isincreased compared to the medium control as shown in FIG. 10b,c. Incontrast, treatment with TRAIL or anti-DR5 antibody is not associatedwith increased release of these MMPs.

EXAMPLE 11A Failure to Induce Hepatocellular Toxicity

[0277] For 24-hour cell viability assays, fresh normal human hepatocytesin 96-well plates were purchased from In Vitro Technology (Baltimore,Md.). The hepatocytes are cultured in the Hepatocyte Culture Mediumcontaining 1 μg/ml of either soluble TRAIL or TRA-8. For 6-hourviability assays, normal hepatocytes or hepatocellular cancer cells areisolated from fresh surgical specimens collected from UAB TissueProcurement Center. All reagents for isolation of human hepatocytesincluding hepatocytes perfusion buffer, digest medium, washing medium,and attachment medium were purchased from Gibco. The tissue slides aredigested in the Hepatocyte Digest Medium at 37° C. with shaking (50 rpm)for one hour. The isolated hepatocytes are harvested by low speedcentrifugation (50 g, 3 min), and washed with the Hepatocyte WashingMedium six times. Single cell suspension of hepatocytes are cultured inthe Attachment Medium containing 10% FCS in 96-well Matrigel plates (BD)for six hours. Non-attached hepatocytes are removed by twice washingwith pre-warmed attachment medium. Attached hepatocytes are furtherincubated with various concentrations of soluble TRAIL or FasL in thepresence of crosslinker, or TRA-8 or CH11 for 6 hours.

[0278] TRAIL has at least two receptors (DR4 and DR5) that are capableof inducing apoptosis. TRA-8 is used to determine whether crosslinkingof DR5 alone is sufficient to induce apoptosis of normal hepatocytes.DR5 expression at the protein level is examined initially in five normalhuman liver tissues and five liver cancer tissues by in situimmunohistochemistry using TRA-8. Sections from the normal liver tissuesshowed normal architecture and cell morphology on H&E staining (FIG.11a, left upper panels) in the absence of positive reactivity with TRA-8for DR5 (FIG. 11a, left lower panels). In contrast, the humanhepatocellular carcinoma tissue reacted positively with TRA-8 in apattern consistent with both cell membrane and cytoplasmic presence ofDR5 on the cancerous cells. The human hepatocellular carcinoma cell lineHepG2 is also positive for DR5. These results are consistent among thefive normal liver tissues, and only one (liver adenoma) out of fiveliver cancer tissues is DR5-negative. These results are consistent withthe Western blot data, shown in FIG. 5a, that, as with other normaltissues, normal human liver tissue does not express significant levelsof DR5 protein. Furthermore, Western blot analysis of isolated, normalhuman hepatocytes probed with TRA-8 does not reveal detectable levels ofDR5.

[0279] Cell surface expression of DR5 on human hepatocytes by flowcytometry analysis demonstrated that freshly prepared normal hepatocytesdid not express detectable levels of cell surface DR5 (FIG. 11b, topleft panels). Neither is it detected on normal human hepatocytes thathad been cryopreserved or placed in short-term culture. In contrast,freshly isolated hepatocellular carcinoma cells as well as HepG2 cellsexpress cell surface DR5. Using Fas as a comparison, the normalhepatocytes, hepatocellular carcinoma cells, and HepG2 cells allexpressed equivalent levels of Fas (FIG. 11b, lower panels). Theseresults are consistent with those obtained using in situimmunohistochemistry and Western blot and indicate that cell surface DR5is highly expressed in cancerous liver cells but not normal hepatocytes.The presence of mRNA levels for DR4, DR5, DcR1 and DcR2 in humanhepatocytes, demonstrated by RT-PCR²³, suggests that human hepatocytesmight express very low levels of DR5 protein that are below thethreshold for detection by TRA-8.

[0280] To determine whether TRA-8 induces hepatocellular toxicity, thesusceptibility of normal human hepatocytes to apoptosis induced by TRA-8and by soluble TRAIL plus crosslinker is examined. When normalhepatocytes are cultured in the presence of a high concentration ofTRAIL, a time-dependent decrease in cell viability is observed byATPLite (FIG. 12a) and MTT assays. TRAIL-mediated cell death of normalhepatocytes could be seen as early as four hours after addition ofTRAIL. At end of a 24-hour culture, more than 80% of the hepatocytes arekilled by TRAIL. In contrast, during the same culture period, TRA-8 didnot induce significant cell death in normal hepatocytes. The condensednuclei stained with Hoechst, a characteristic of apoptosis, areincreased in TRAIL-treated but not TRA-8-treated hepatocytes (FIG. 12b).The number of apoptotic hepatocytes is well correlated with decreasedcell viability as determined by ATPLite assay, suggesting thatTRAIL-induced cell death of hepatocytes is mediated by apoptosis. Thisis confirmed by the ability of Z-VAD to inhibit TRAIL-mediated toxicityof hepatocytes. As cycloheximide is a potent apoptosis enhancer, theeffect of this compound on TRAIL and TRA-8-treated hepatocytes isinvestigated. During a four-hour culture, cycloheximide significantlyenhanced the cell death of hepatocytes induced by TRAIL, with greaterthan 70% hepatocytes being killed by TRAIL in the presence ofcycloheximide (FIG. 12c). However, cycloheximide treatment is unable toenhance TRA-8-mediated cell death in hepatocytes. To compare thecharacteristics of apoptosis induced by TRA-8 with that induced by TRAILin hepatocytes, normal hepatocytes as well as cancer cells are incubatedwith variable concentrations of soluble TRAIL with crosslinker or TRA-8.During a 6-hour culture period, TRAIL induced a moderate apoptoticresponse in normal hepatocytes. Over 20% of hepatocytes are killed inthe presence of 500 ng/ml TRAIL (FIG. 12d, upper left). TRA-8-treatmentof normal hepatocytes did not elicit any significant cell death over thesame time period. In contrast to normal hepatocytes, primaryhepatocellular carcinoma cells (FIG. 12d, upper middle) and HepG2 cells(FIG. 12d, upper right) are highly susceptible to apoptosis mediated byeither TRAIL or TRA-8. Over 80% of hepatocellular carcinoma cells andnearly 100% of HepG2 cells are killed during the 8-hour culture period.These results indicate that normal hepatocytes are completely resistantto TRA-8-mediated apoptosis, and are much less susceptible toTRAIL-mediated apoptosis than are liver cancer cells. Using Fas ligandand anti-Fas antibody (CH-11), there is no significant difference in thesusceptibility to Fas-mediated apoptosis among normal hepatocytes,hepatocellular carcinoma cells, and HepG2 cells (FIG. 12d, lowerpanels).

COMPARATIVE EXAMPLE 11B Human Membrane-Bound TRAIL Induction ofHepatitis in vivo

[0281] 8-10 week-old female B6 mice are intravenously injected with 109pfu of Ad/hTRAIL with the equal number of Ad/Tet-on. Mice are fed withdifferent concentrations of tetracycline in their drinking waterimmediately after inoculation of adenoviral vectors. Liver injury isdetermined by serum levels of AST using an AST diagnostic kit (Sigma).Expression of TRAIL is determined by Northern blot analysis.

[0282] To determine whether the membrane bound form of TRAIL inducesliver damage in vivo, a recombinant adenoviral vector encoding the fulllength human TRAIL (Ad/hTRAIL) is constructed, the expression of whichis under the control of the tetracycline-inducible promoter. Twenty-fourhours after intravenous inoculation of B6 mice with Ad/hTRAIL,tetracycline-induced expression of human TRAIL is observed in the liverin a dose-dependent fashion as demonstrated by Northern blot analysis(FIG. 13a). The expression levels of TRAIL correlated well with liverdamage as shown by a tetracycline-dependent increase in serum levels oftransaminases, again in a dose-dependent fashion (FIG. 13b). As theinoculation with adenoviral vector per se might increase thesusceptibility of hepatocytes to TRAIL-mediated apoptosis, thehepatocytes from mice inoculated with Ad/TRAIL are isolated and testedfor TRAIL-mediated cell death. There is no significantly increased celldeath of Ad/TRAIL infected hepatocytes compared to those from controlmice (FIG. 13c, left panel). Moreover, Ad/TRAIL inoculated mice did notexhibit increased liver injury after intravenous injection of solublehuman TRAIL. Thus, it follows that hepatitis induced by Ad/TRAIL ismediated by high levels of TRAIL expression in its membrane form.Histologic analysis of liver sections revealed that damage to thehepatocytes is apparent as early as 24 hours after vector inoculation(FIG. 13d), and persisted for at least 7 days (FIG. 13e). Thesepathologic alterations in the liver also are tetracycline-dependent andoccurred in a dose-dependent manner. The early phase, within 24 hours oftreatment, of TRAIL-induced liver damage is characterized by foci ofnecrosis. Infiltration of inflammatory cells is not observed at thisstage, but hemorrhage had occurred. By day 7 after inoculation, diffuseliver damage is apparent with marked lobular disarray, severedegeneration of hepatocytes with irregularly clumped cytoplasm and largeclear spaces, and prominent apoptosis and necrosis. An extensiveinfiltrate of mononuclear cells is a characteristic feature at thisstage. These results indicate that human TRAIL in its membrane-boundform is able to induce liver damage in vivo. Despite the propensity ofhuman TRAIL to cause severe hepatitis in mice, it did not induce alethal response. In contrast, mice inoculated with similartetracycline-controlled vectors encoding Fas ligand developed fulminanthepatitis with massive apoptosis and necrosis of hepatocytes accompaniedby severe hemorrhage and by mortality occurring in a tetracyclinedose-dependent within 72 hours of inoculation. The mortality ratereached 100% within 48 hours in those subgroups receiving 3 mg/ml ormore of tetracycline. In contrast, all of the mice that receivedAd/hTRAIL, regardless of the dose of tetracycline, are still alive fourweeks after inoculation. Thus, it follows that, in vivo, themembrane-bound form of TRAIL is a less potent inducer of hepatocellulardamage than Fas ligand. They further suggest that TRAIL might induceliver damage through a mechanism that differs from the mechanismunderlying the toxicity of Fas ligand.

EXAMPLE 12 Activated Human T and B Cells Express Increased Levels of DR5

[0283] To determine whether DR5 plays a role in TRAIL-mediated apoptosisof activated T cells and B cells, surface expression of DR5 on restingand activated T and B cells using TRA-8 is examined. The unstimulatedhuman T cells in PBMC did not express significant levels of DR5 (FIG.14). At 48 hours after either anti-CD3 or Con-A stimulation, cellsurface DR5 expression is significantly increased. Similarly, theunstimulated B cells expressed very low levels of DR5. Stimulation withanti-μ but not LPS resulted in increased cell surface expression of DR5.These results indicate that both activated T and B cells express higherlevels of cell surface DR5. Cells are stained with 20 μg/ml TRA-8 and PEanti-mouse IgG1.

EXAMPLE 13 Activated T and B Cells Become Susceptible to TRA-8 MediatedApoptosis

[0284] To determine whether activated T and B cells are susceptible toTRA-8-mediated apoptosis, the T cells and B cells of human PBMC arestimulated with anti-CD3 or anti-μ in vitro for 48 hours, respectively.The viable cells and proliferating blast cells are collected by gradientcentrifugation, and incubated with various concentrations of TRA-8.Unstimulated T cells and B cells are not susceptible to TRA-8-mediatedapoptosis (FIG. 15). Total stimulated T cells and B cells showed amoderately increased susceptibility to TRA-8-mediated apoptosis, with20% cells being killed by TRA-8 after overnight culture. The highlyproliferating blast T cells are even more susceptible to TRA-8 mediatedapoptosis. More than 70% of the blast T cells could be killed by TRA-8.The blast B cells are also more susceptible to TRA-8 mediated apoptosiscompared to others. These results indicate that activated T and B cellsare susceptible to DR5-mediated apoptosis.

EXAMPLE 14 TRA-8 Depletes Activated T Cells in Human/SCID Mice

[0285] To determine the in vivo anti-T cell efficacy of TRA-8, NOD/SCIDmice are intravenously injected with 1×10⁸ human PBMC. Normally, thehuman T cells in SCID mice are quickly activated in response toxenogeneic stimulation. The human PBMC/SCID mice are intraperitoneallyinjected with 100 μg TRA-8 or control IgG1 from the day of transfer,repeated daily for three days. Five days after transfer, the mononuclearcells are isolated from the spleen and stained with anti-human CD3antibody, and the lymphocyte population is gated by flow cytometryanalysis, and CD3 positive human T cells are analyzed. Approximately 30%of splenic lymphocytes are human T cells as determined by anti-human CD3staining in control treated mice. However, only a few human T cells(less than 3%) are observed among the splenic lymphocytes in TRA-8treated mice (FIG. 16). In situ histological study revealed that in thespleen of control mice, the human T cells are repopulated in the spleenwith only a few apoptotic cells observed as demonstrated by TUNELstaining. In contrast, repopulation with viable human T cells is notobserved in the spleen of TRA-8 treated mice, rather many apoptoticcells are observed (FIG. 17). These results demonstrate that TRA-8 hasanti-T cell activity in vivo, and indicate the utility of the inventiveantibodies for the treatment of GVH disease.

EXAMPLE 15 Anti-Cancer Therapeutic Activity of TRA-8

[0286] 15.1 DR5 Expression and Function in Human Cancer Tissues and CellLines

[0287] i) DR5 expression in human cancer tissues by in situ stainingwith TRA-8. To determine whether cancer cells and tissues differentiallyexpress higher levels of DR5, a panel of human cancer tissues includingover 20 breast cancers, 6 ovarian cancers, 5 colon cancers and 5prostate cancers are stained with TRA-8 for immunohistochemistry. Themajority of these cancer tissues expressed detectable DR5. Theexpression levels of DR5 in these cancer tissues varied. In general,cancer tissues expressed higher levels of DR5 than uninvolved tissues.In addition, DR5 expression is apparently not correlated with themutation of p53.

[0288] ii) DR5 expression and function in human cancer cell lines (Table2). Nine human breast cancer cell lines, three ovarian cancer lines,three colon cancer lines and three prostate cancer lines are examinedfor cell surface expression of DR5 and susceptibility to TRA-8-inducedapoptosis in vitro. 7 of 9 breast cancer lines, 3 of 3 ovarian cancerlines, 3 of 3 colon cancer lines and 3 of 3 prostate cancer linesexpressed variable levels of cell surface DR5. Of 9 breast cancer lines,three are very susceptible, three are intermediate and three areresistant to TRA-8-mediated apoptosis. All three ovarian cancer linesare very susceptible. One of three colon cancer lines is verysusceptible, while two have intermediate sensitivity. Two of threeprostate cancer lines have intermediate sensitivity and one isresistant. TABLE 2 Expression and function of DR5 in human cancer cells.Cell line Origin Expression¹ Susceptibility² 2LMP breast + ++++ LCC6breast +++ ++++ MB468 breast +++ +++ MB231 breast ++ +++ ZR-75-1 breast+++ ++ SKBR3 breast + ++ MB453 breast ++ + BT474 breast + − DY36T2breast − − Caov-3 ovary + ++++ OVCAR-3 ovary ++ ++++ Skov-3 ovary + +++WiDR colon +++ ++++ HST29 colon ++ +++ T84 colon + ++ PC3 prostate +++++ LnCap prostate +++ + Du-145 prostate +++ +

[0289] iii) Combined cytotoxicity of TRA-8 with adriamycin. In severalbreast cancer lines, the effect of adriamycin on TRA8-induced apoptosisis examined. High doses of adriamycin exhibited an additive effect.However, in some of TRA-8 resistant lines, low doses of adriamycinsynergistically enhance TRA-8-induced apoptosis.

[0290] iv) In vitro and in vivo binding activity of TRA-8 to humancancer cells. Using radioisotope labeled TRA-8. The binding activity ofTRA-8 to a breast cancer line is examined in vitro and in vivo in SCIDmice implanted with tumor. The in vitro binding activity to cancer cellsis estimated as a Kd value of 3 nM, which is constant with our previousestimation using ELISA, and at least 50-fold higher than soluble TRAIL.In vivo, TRA-8 localized to implanted tumor tissues.

[0291] 15.2. Therapy of Chronic Lympholytic Leukemia in NOD/SCID Micewith TRA-8

[0292] Chronic lympholytic leukemia (CLL) is a common form of B cellmalignancy. Most malignant B cells in CLL are of the mature phenotypeand are resistant to many apoptosis stimuli. DR5 expression and functionin the B cells of five patients with CLL is examined. All patients hadhigh counts of peripheral B cells as shown by more than 95% CD19+B cellsin PBMC. Compared to normal primary B cells, the CLL B cells of allpatients had higher levels of cell surface DR5 and are more susceptibleto TRA-8 induced apoptosis in vitro. Interestingly, the CLL B cells arealso sensitive to bisindolemaleimide VIII (BisVIII) inducedcytotoxicity. Following combined treatment with TRA-8 and BisVIII,nearly 50% of CLL B cells are killed while normal B cells remainedunresponsive (FIG. 18). Transfer of CLL B cells into NOD/SCID miceresulted in about 25%-30% CD19+B cells repopulated in the spleen ofrecipient mice at five days after transfer. However, three doses of 100μg TRA-8 treatment completely eliminated CLL B cells of four out of fivepatients in the spleen of the recipient SCID mice. Thus, TRA-8 alone orin concert with other substances is active as a therapeutic agent forchronic lympholytic leukemia.

EXAMPLE 16 cDNA Cloning

[0293] (1) Determination of the N-Terminal Amino Acid Sequences of theHeavy and Light Chains of TRA-8

[0294] In order to obtain cDNAs of the heavy and light chains of TRA-8,the N-terminal amino acid sequences of the heavy and light chains ofTRA-8 and cloned TRA-8 genes are determined by known techniques.

[0295] Ten μg of the solution containing the anti-human DR5 antibodyTRA-8 is subjected to SDS-polyacrylamide gel electrophoresis(“SDS-PAGE”), using a gel concentration of 12% w/v, 100 V constantvoltage, for 120 minutes. After electrophoresis, the gel is immersed intransfer buffer 25 mM Tris-HCl (pH 9.5), 20% methanol, 0.02% v/v SDS for5 minutes. After this time, the protein content of the gel istransferred to a polyvinylidene difluoride membrane (“PVDF membrane”;pore size 0.45 um; Millipore, Japan), presoaked in transfer buffer,using a blotting apparatus (KS-8451; Marysol) under conditions of 10 Vconstant voltage, 4° C., for 14 hours.

[0296] After this time, the PVDF membrane is washed with washing buffer25 mM NaCl, 10 mM sodium borate buffer (pH 8.0), then stained in astaining solution (50% v/v methanol, 20% v/v acetic acid and 0.05% w/vCoomassie Brilliant Blue) for 5 minutes to locate the protein bands. ThePVDF membrane is then destained with 90% v/v aqueous methanol, and thebands corresponding to the heavy chain, the band with the lower mobilityand light chain, the band with the higher mobility previously located onthe PDVF membrane are excised and washed with deionized water.

[0297] The N-terminal amino acid sequence of the heavy and light chainsare determined by the Edman automated method (Edman, P., et al., (1967),Eur. J. Biochem., 1, 80) using a gas-phase protein sequencer (PPSQ-10;Shimadzu Seisakusyo, K. K.).

[0298] The N-terminal amino acid sequence of the band corresponding tothe heavy chain is determined to be:

[0299]Glu-Val-Met-Leu-Val-Glu-Ser-Gly-Gly-Gly-Leu-Val-Lys-Pro-Gly-Gly-Ser-Leu-Lys-Leu(SEQ ID No. 4 of the Sequence Listing);

[0300] and that of the band corresponding to the light chain isdetermined to be:

[0301]Asp-Ile-Val-Met-Thr-Gln-Ser-His-Lys-Phe-Met-Ser-Thr-Ser-Val-Gly-Asp-Arg-Val-Ser(SEQ ID No. 5 of the Sequence Listing).

[0302] Comparison of these amino acid sequences with the database ofamino acid sequence of antibodies produced by Kabat et al. (Kabat E. A.,et al., (1991), in “Sequences of Proteins of Immunological Interest Vol.II,” U.S. Department of Health and Human Services) revealed that theheavy chain (y1 chain) and the light chain (k chain) of TRA-8 belongedto subtypes 3d and 1, respectively.

[0303] (2) cDNA cloning

[0304] Based on above findings, oligonucleotide primers are synthesizedwhich would be expected to hybridize with portions of the5′-untranslated regions and the very ends of the 3′-translated regionsof the genes belonging to these mouse subtypes. Then, cDNAs encoding theheavy and light chains of TRA-8 are cloned by the following combinationof reverse transcription and PCR (RT-PCR):

[0305] a) Template

[0306] The total RNA of TRA-8 hybridoma (ATCC No. PTA-1428) is extractedby using TRIzol Reagent (GIBCO BRL). The template for the PCR reactionused cDNA that is obtained by using the First-Strand cDNA synthesis kit(Amersham Pharmacia Biotech) according to the instruction manualprovided with the kit.

[0307] b) PCR Primers

[0308] The following oligonucleotide primers are synthesized for thePCR: 5′-cagcactgaa cacggacccc-3′; (H5NCS1: SEQ ID No. 6 of the SequenceListing) 5′-aaaggtaatt tattgagaag-3′; (H5NCS2: SEQ ID No. 7 of theSequence Listing) 5′-cctcaccatg aacttcgggc-3′; (H5SS1: SEQ ID No. 8 ofthe Sequence Listing) 5′-ctgttgtatg cacatgagac-3′; (H5SS2: SEQ ID No. 9of the Sequence Listing) 5′-gaagtgatgc tggtggagtc-3′; (H5CS1: SEQ ID No.10 of the Sequence Listing) 5′-agtgtgaagt gatgctggtg-3′; (H5CS2: SEQ IDNo. 11 of the Sequence Listing) 5′-tttaccagga gagtgggaga g-3′; (H3CR:SEQ ID No. 12 of the Sequence Listing) 5′-tgcagagaca gtgaccagag-3′;(H3VR: SEQ ID No. 13 of the Sequence Listing) 5′-tgttcaggaccagcatgggc-3′; (L5NCS1: SEQ ID No. 14 of the Sequence Listing)5′-aagacatttt ggattctaac-3′; (L5NCS2: SEQ ID No. 15 of the SequenceListing) 5′-tatcatgaag tctttgtatg-3′; (L5SS1: SEQ ID No. 16 of theSequence Listing) 5′-gatggagaca cattctcagg-3′; (L5SS2: SEQ ID No. 17 ofthe Sequence Listing) 5′-gacattgtga tgacccagtc-3′; (L5CS: SEQ ID No. 18of the Sequence Listing) 5′-ttaacactca ttcctgttga-3′; (L3CR: SEQ ID No.19 of the Sequence Listing) and 5′-gactgggtca tcacaatgtc-3′. (LCSR: SEQID No. 20 of the Sequence Listing)

[0309] Unless otherwise specified, all oligonucleotides in theseExamples are synthesized by Pharmacia Biotech. All oligonucleotides arestored at −20° C. after being dissolved in distilled water.

[0310] c) PCR Reaction

[0311] Composition of the PCR reaction solution:

[0312] template cDNA, 5 μl of total 33 μl reaction

[0313] primer DR5p1, 10 pmol;

[0314] primer DR5p2, 10 pmol;

[0315] 10×concentrated PCR buffer (provided with the kit), 10 μl;

[0316] dNTPs (each 2.5 mM), 4 μl; and

[0317] Taq polymerase (Promega), 5 units.

[0318] Sterile distilled water is added to the solution to a totalvolume of 100 μl. Unless otherwise specified, dNTPs are an equimolarmixture of DATP, dCTP, dGTP and dTTP (2.5 mM each).

[0319] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 2 minutes, after which a cycle of heating to 94° C.for 30 sec, 52° C. for 1 minute and 72° C. for 3 minutes, is repeated 40times. After completion of this procedure, the reaction solution isheated at 72° C. for 10 minutes.

[0320] The amplified DNA fragments, thus obtained, are separated on a 1%agarose gel containing 0.25 μg/ml ethidium bromide. The bands determinedto contain the desired DNA fragments are cut out using a razor blade andthe DNA is recovered therefrom using the Gene Clean kit (BIO101). TheDNA fragment is cloned using pGEM-T Easy vector (Promega). This isperformed as follows.

[0321] The DNA fragment recovered from the PCR reaction solution,together with 50 ng of pGEM-T Easy vector (provided with the kit), ismixed with 1 μl of 10×ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6mM magnesium chloride, 5 mM sodium chloride, 7 mM P-mercaptoethanol, 0.1mM ATP, 2 mM DTT, 1 mM spermidine, and 0.1 mg/ml bovine serum albumin),to which 4 units of T4 DNA ligase (1 μl) has been added. The totalvolume of the mixture is adjusted to 10 μl with sterile deionized water,and the resulting ligase solution is incubated at 14° C. for 15 hours.After this time, 2 μl of the ligase reaction solution is added to 50 μlof competent E. coli strain JM109 (provided with the kit and brought tocompetence in accordance with the instruction manual) to which 2 μl of0.5 M P-mercaptoethanol had been added, and the resulting mixture iskept on ice for 30 minutes, then at 42° C. for 30 seconds, and again onice for 5 minutes. Next, 500 μl of medium containing 2% v/v tryptone,0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM potassiumchloride, 1 mM magnesium chloride, and 20 mM glucose (hereinafterreferred to as “SOC” medium) is added to the culture, and the mixture isincubated for 1 hour at 37° C. with shaking. After this time, theculture is spread on an L-broth agar plate (1% v/v tryptone, 0.5% w/vyeast extract, 0.5% w/v sodium chloride, 0.1% w/v glucose, and 0.6% w/vbacto-agar (Difco)), containing 100 μg/ml. Ampicillin resistant coloniesappearing on the plate are selected and scraped off with a platinumtransfer loop, and cultured in L-broth medium containing 100 μg/mlampicillin at 37° C., overnight, with shaking at 200 r.p.m. Afterincubation, the cells are harvested by centrifugation, from whichplasmid DNA is prepared by the alkali method. The obtained plasmid isdesignated as plasmid pH 62 for heavy chain of TRA-8 or pL28 for lightchain of TRA-8. The transformant E coli strains harboring these plasmid,designated as E. coli JM109/pH 62 and E. coli JM109/pL28 were depositedwith International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, 1-1, Higashi 1 chomeTsukuba-shi, Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, inaccordance with the Budapest Treaty for the Deposit of Microorganisms,and were accorded the accession numbers FERM BP-7560 and FERM BP-7561,respectively. The nucleotide sequences of these DNAs encoding the heavychain and the light chain of TRA-8 are confirmed by the dideoxy method(Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin Elmer AppliedBiosystems, Japan).

[0322] The nucleotide sequences of the heavy and light chains of TRA-8are given as SEQ ID No. 21 and No. 22 of the Sequence Listing,respectively. The amino acid sequences of the heavy and light chains ofTRA-8 are given as SEQ ID No. 23 and No. 24 of the Sequence Listing,respectively. The N-terminal amino acid sequences of the heavy and lightchains of TRA-8 established in above matched perfectly. Furthermore,when the amino acid sequences of the heavy and light chains are comparedwith the database of amino acid sequences of antibodies, it isestablished that, for the heavy chain, nucleotide Nos. 58 to 414 in SEQID No. 21 constituted the variable region, while nucleotide Nos. 415 to1392 in SEQ ID No. 21 constituted the constant region. For the lightchain, nucleotide Nos. 64 to 387 in SEQ ID No. 22 constituted thevariable region, while nucleotide Nos. 388 to 702 in SEQ ID No. 22constituted the constant region. The locations and sequences of the CDRsare also elucidated by comparing the homologies with the database. Theamino acid sequences of CDR1, CDR2, and CDR3 of heavy chain of TRA-8 areshown in SEQ ID No. 25, No. 26, and No. 27, respectively. The amino acidsequences of CDR1, CDR2, and CDR3 of light chain of TRA-8 are shown inSEQ ID No. 28, No. 29, and No. 30, respectively.

EXAMPLE 17 Designing a Humanized Version of the TRA-8 Antibody

[0323] (1) Molecular Modeling of a Variable Region of TRA-8

[0324] Molecular modeling of the variable region of TRA-8 is performedby the method generally known as homology modeling (Methods inEnzymology, 203, 121-153, (1991)). The primary sequences of variableregions of human immunoglobulin registered in the Protein Data Bank(Nuc. Acid Res. 28, 235-242 (2000)), for which the three-dimensionalstructures derived from x-ray crystallography are available, arecompared with the framework regions of TRA-8 determined above. As aresult, 1NCD and 1HIL are selected as having the highest sequencehomologies to the framework regions for the light and heavy chains ofTRA-8, respectively. Three-dimensional structures of the frameworkregions are generated by combining the coordinates of 1NCD and 1HILwhich correspond to the light and heavy chains of TRA-8, to obtain the“framework model”. Using the classification defined by Chothia et al.,the CDRs of TRA-8 are classified as follows; CDRL₁, CDRL₂, CDRH₁ andCDRH₂ belong to canonical classes 2,1,1,3, respectively, while CDRL₃does not belong to any specific canonical classes. The CDR loops ofCDRL₁, CDRL₂, CDRH₁, CDRH₂ are fixed to the conformations inherent totheir respective canonical classes, and integrated into the frameworkmodel. CDRL₃ is assigned the conformation of cluster 8A, according tothe classification of Thornton et al. (J. Mol. Biol., 263, 800-815,(1996)), and CDRH₃ is classified into k(8)C using the H3 rule (FEBSletter 455,188-197(1999)). Then representative conformations for CDRL₃and CDRH₃ are integrated into the framework model.

[0325] Finally, energy calculations are carried out to eliminateunfavorable inter-atomic contacts, in order to obtain a probablemolecular model of TRA-8's variable region in terms of energy. The aboveprocedure is performed using the commercially available common molecularmodeling system ABM (Oxford Molecular Limited, Inc.). For the molecularmodel obtained, the accuracy of the structure is further evaluated usingthe software, PROCHECK (J. Appl. Cryst. (1993), 26, 283-291).

[0326] (2) Designing the Amino Acid Sequences for Humanized TRA-8.

[0327] Construction of humanized TRA-8 antibodies is performed by themethod generally known as CDR grafting (Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989)). The acceptor antibody is chosen based on the aminoacid homology in the framework region. The sequences of framework regionin TRA-8 are compared with all the human framework sequences in theKabat database of amino acid sequences of antibodies (Nuc. Acid Res. 29,205-206 (2001)). As a result, mAB58′ CL antibody is selected as anacceptor due to the highest sequence homology of 80% for the frameworkregion. The amino acid residues in the framework region for mAb58′CL arealigned with that for TRA-8 and the positions where different aminoacids are used are identified. The location of those residues areanalyzed using the three dimensional model of TRA-8 constructed aboveand the donor residues which should be grafted on the acceptor arechosen by the criteria given by Queen et al. (Proc. Natl. Acad. Sci. USA86, 10029-10033 (1989)). Humanized TRA-8 sequences are constructed asdescribed in the following example by transferring several donorresidues into acceptor antibody, mAb58′CL.

EXAMPLE 18 Construction of an Expression Vector for the Heavy Chain ofthe Humanized Antibody

[0328] (1) Construction of Plasmid Carrying the Heavy Chain VariableRegion DNA of Humanized TRA-8

[0329] In order to determine the activity of humanized TRA-8, theplasmid carrying the heavy chain of humanized TRA-8 is constructed asfollows. However, it is appreciated the humanization of TRA-8 is notlimited to these examples.

[0330] As shown in SEQ ID No. 31 of the Sequence Listing, humanizationof the amino acid sequences of the heavy chain of the mouse anti-humanDR5 antibody TRA-8 entailed replacing the 13th amino acid (lysine), the19th amino acid (lysine), the 40th amino acid (threonine), the 42ndamino acid (glutamic acid), the 44th amino acid (arginine), the 84thamino acid (serine), the 88th amino acid (serine), the 93rd amino acid(methionine), the 114th amino acid (threonine), the 115th amino acid(leucine) with glutamine, arginine, alanine, glycine, glycine,asparagine, alanine, valine, leucine, and valine, respectively.

[0331] The plasmid carrying DNA encoding heavy chain variable region ofhumanized TRA-8 (SEQ ID No. 31 of the Sequence Listing) are constructedas follows.

[0332] PCR is used to construct the following DNA sequences, each ofwhich comprised described above:

[0333] The following 12 oligonucleotides are synthesized: 5′-ttggataagcttggcttgac ctcaccatgg gatggagctg tatcatcctc ttcttggtag (A; SEQ ID No.32) caacagctac aggtgtccac-3′; 5′-tctgaagtaa tgctggtgga gtctgggggaggcttagtac agcctggagg gtccctgaga (B; SEQ ID No. 33) ctctcctgtgcagcctctgg-3′; 5′-attcactttc agtagttatg taatgtcttg ggttcggcag gcaccagggaagggtctgga (C; SEQ ID No. 34) gtgggttgca accattagta-3′; 5′-gtggtggtagttacacctac tatccagaca gtgtgaaggg ccgattcacc atctccagag (D; SEQ ID No.35) acaatgccaa gaacaccctg-3′; 5′-tatctgcaaa tgaacagtct gagagcagaggacacggctg tttattactg tgcaagaagg (E; SEQ ID No. 36) ggtgactctatgattacgac-3′; 5′-ggactactgg ggccaaggga ccctggtcac agtctcctca gcctc caccaagggcccat (F; SEQ ID No. 37) cggtc-3′; 5′-ctaccaagaa gaggatgatacagctccatc ccatggtgag gtcaagccaa gcttatccaa-3′; (G; SEQ ID No. 38)5′-tctcagggac cctccaggct gtactaagcc tcccccagac tccaccagca ttacttcaga (H;SEQ ID No. 39) gtggacacct gtagctgttg-3′; 5′-tccagaccct tccctggtgcctgccgaacc caagacatta cataactact gaaagtgaat (I; SEQ ID No. 40)ccagaggctg cacaggagag-3′; 5′-ctctggagat ggtgaatcgg cccttcacac tgtctggatagtaggtgtaa ctaccaccac (J; SEQ ID No. 41) tactaatggt tgcaacccac-3′;5′-ccttcttgca cagtaataaa cagccgtgtc ctctgctctc agactgttca tttgcagata (K;SEQ ID No. 42) cagggtgttc ttggcattgt-3′; and 5′-gaccgatggg cccttggtggaggctgagga gactgtgacc agggtccctt ggccccagta (L; SEQ ID No. 43)gtccgtcgta atcatagagt cacc-3′. The following 2 PCR primers aresynthesized as described above: 5′-ttggataagc ttggcttgac-3′; and (P1;SEQ ID No. 44) 5′-gaccgatggg cccttggtgg a-3′. (P2; SEQ ID No. 45)

[0334] The synthesis of DNA encoding a polypeptide chain comprising asecretion signal sequence, a variable region of humanized TRA-8 heavychain and the 8 amino acid residues at the N-terminus of the IgG-CH1region is performed using a combination of PCR respectively.

[0335] The DNA fragment is prepared as follows. Composition of the PCRreaction solution:

[0336] oligonucleotide A, 10 pmol;

[0337] oligonucleotide B, 10 pmol;

[0338] oligonucleotide C, 10 pmol;

[0339] oligonucleotide D, 10 pmol;

[0340] oligonucleotide E, 10 pmol;

[0341] oligonucleotide F, 10 pmol;

[0342] oligonucleotide G, 10 pmol;

[0343] oligonucleotide H, 10 pmol;

[0344] oligonucleotide I, 10 pmol;

[0345] oligonucleotide J, 10 pmol;

[0346] oligonucleotide K, 10 pmol;

[0347] oligonucleotide L, 10 pmol;

[0348] oligonucleotide primer P1, 2 μM;

[0349] oligonucleotide primer P2, 2 μM;

[0350] 10×Pyrobest buffer II, 10 μl;

[0351] dNTP mix, 8 μl;

[0352] Pyrobest DNA polymerase, 0.5 μl; and

[0353] Redistilled water to a final volume of 50 μl.

[0354] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 5 minutes, after which a cycle of heating to 98° C.for 10 second, 55° C. for 30 second and 72° C. for 1 minute, is repeated7 times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

[0355] An equal volume of phenol-chloroform (50% v/v phenol saturatedwith water, 48% v/v chloroform, 2% v/v isoamyl alcohol) is added to 200μl of each of the PCR products, and vigorously mixed for 1 minute. Afterthis time, the mixture is centrifuged at 10,000×g, and the aqueous layeris recovered and mixed with an equal volume of chloroform-isoamylalcohol (96% v/v chloroform and 4% v/v isoamyl alcohol), which is againvigorously at 10,000×g and the aqueous layer is recovered. The series ofsteps recited in this paragraph is referred to, hereafter, as “phenolextraction”).

[0356] Ethanol precipitation is then performed on the recovered aqueouslayer. As used and referred to herein, “ethanol precipitation” consistsof adding, with mixing, a one tenth volume of 3M sodium acetate (pH 5.2)and 2.5 volumes of 100% ethanol to the solution to be treated, andfreezing the mixture using dry ice. The resulting mixture is thencentrifuged at 10,000×g to recover DNA as a precipitate.

[0357] After phenol extraction and ethanol precipitation, the resultingDNA precipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandcorresponding to humanized TRA-8 DNA is cut out using a razor blade andeluted from the gel using Geneclean Spin Kit (BIO 101, CA, USA). Afterphenol extraction, the eluted DNA is then concentrated by centrifugationat 7,500×g, followed by ethanol precipitation, and finally dissolved in5 μl of distilled water.

[0358] The resulting, each extracted DNA is cloned using pGEM-T Easyvector (Promega) as follows:

[0359] The DNA fragment recovered from the PCR reaction, 5 μl;

[0360] 10×Taq polymerase buffer, 1 μl;

[0361] dNTP mixture, 1 μl

[0362] Taq polymerase (5 unit/ml), 1 μl; and

[0363] redistilled water to a final volume of 10 μl.

[0364] After the above each solution is reacted at 70° C. for 30minutes, each DNA solution and pGEM-T Easy vector are ligated using aDNA Ligation Kit Version 2.0 (Takara Shuzo Co., Ltd.) using themanufacturer's protocol.

[0365] After 4 hours incubation at 15° C., 2 μl of the incubatedreaction solution is mixed with 100 μl of competent E. coli strain JM109at a cell density of 1-2×10⁹ cells/ml (Takara Shuzo Co., Ltd.), and themixture is kept on ice for 30 minutes, then at 42° C. for 30 seconds,and again on ice for 1 minutes. Then, 500 μl of SOC medium (2% v/vtryptone, 0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM w/vpotassium chloride, 1 mM magnesium chloride, and 20 mM glucose) is addedthe mixture, which is incubated for a further hour, with shaking.Transformant strains are then isolated, and plasmid DNA is prepared fromthe strains as described in “Molecular Cloning A Laboratory Manual”. Thenucleotide sequences of these DNAs encoding the heavy chain of humanizedTRA-8 are confirmed by the dideoxy method (Sanger, F. S., et al.,(1977), Proc. Natl. Acad. Sci. USA, 74:5463-5467) using 3700 DNAAnalyzer (ABI PRISM; Perkin Elmer Applied Biosystems, Japan).

[0366] The resulting plasmids are designated pHB14 (the plasmid carryingcDNA encoding the heavy chain of humanized TRA-8). The transformant EColi strain harboring these plasmid, designated as E. coli JM109/pHB14was deposited with International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, 1-1, Higashi 1chome Tsukuba-shi, Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, inaccordance with the Budapest Treaty for the Deposit of Microorganisms,and was accorded the accession number FERM BP-7556.

[0367] (2) Construction of Expression Plasmids Carrying the Heavy ChainVariable Region DNA of Humanized TRA-8

[0368] Recombinant expression vectors for animal cells are constructedby inserting the DNA encoding the heavy chain of humanized TRA-8 (clonedin above) as follows.

[0369] One μg of plasmid pSRHHH3 (European patent application EP0-909-816-A1) carrying the heavy chain variable region of humanizedanti-Fas monoclonal antibody HFE7A and human IgG1 constant regiongenomic DNA, an expression vector for mammalian cells, is digested withthe restriction enzymes HindIII and ApaI, and separated by 3% agarosegel electrophoresis. After electrophoresis, the gel is stained with a 1μg/ml aqueous solution of ethidium bromide to allow detection of DNAunder UV light. The vector DNA bands containing human IgG1 constantregion genomic DNA without the heavy chain variable region of humanizedHFE7A are cut out using a razor blade and eluted from the gel usingGeneclean Spin Kit (BIO 101, CA, USA). After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater and then dephosphorylated using CIP. The resulting digested,dephosphorylated plasmid (100 ng) is ligated with 1 μg of the pHB14 DNAfragment containing the DNA encoding the heavy chain variable region ofhumanized TRA-8, which had also been digested with HindIII and ApaI,using a DNA Ligation Kit Version 2.0 (Takara Shuzo Co., Ltd.). Theligation mixture is then used to transform E. coli JM109, which is thenplated on LB agar plates containing 50 μg/ml ampicillin.

[0370] The transformants obtained by this method are cultured in 2 ml ofliquid LB medium containing 50 μg/ml ampicillin at 37° C. overnight, andplasmid DNA is subsequently extracted from the resulting culture by thealkaline-SDS method.

[0371] The extracted plasmid DNA is digested with HindIII and ApaI, andsubjected to 3% w/v agarose gel electrophoresis to confirm the presenceor absence of the insert of the DNA encoding the heavy chain variableregion of humanized TRA-8. The insertion and orientation of the desiredDNA fragment in the vector is confirmed by DNA sequencing using a genesequence analyzer (ABI Prism 3700 DNA Analyzer; Applied Biosystems). Theresulting expression plasmid carrying cDNA encoding the heavy chain ofhumanized TRA-8 is designated pHB 14-1.

EXAMPLE 19 Construction of an Expression Vector for the Light Chain ofthe Humanized ANTIBODY

[0372] (1) Construction of Vectors for the Light Chains of HumanizedVersions of TRA-8 Antibody

[0373] As shown in SEQ ID No. 46 of the Sequence Listing, in humanizingthe amino acid sequence of the light chain of the mouse anti-human DR5antibody TRA-8, 8th amino acid (histidine), 9th amino acid (lysine),10th amino acid (phenylalanine), 11th amino acid (methionine), 13thamino acid (threonine), 20th amino acid (serine), 42nd amino acid(glutamine), 43rd (serine), 60th amino acid (aspartic acid), 63rd aminoacid (threonine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 85th aminoacid (aspartic acid), 87th amino acid (phenylalanine),and 99th aminoacid (glycine) 103rd amino acid (leucine) and 108th amino acid (alanine)from the N-terminus of the amino acid sequence of the TRA-8 light chainare replaced with proline, serine, serine, leucine, alanine, threonine,lysine, alanine, serine, serine, serine, leucine, proline,phenylalanine, threonine, tyrosine, glutamine, valine and threoninerespectively. The resulting sequence is designated LM2.

[0374] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 is constructedas follows.

[0375] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized TRA-8

[0376] DNA coding for the LM2 polypeptide chain (SEQ ID No. 46 of theSequence Listing), each of which is a fusion of the variable region ofhumanized anti-DR5 antibody TRA-8 light chain and the constant region ofthe human Ig light chain (K chain), are respectively synthesized byusing combinations of PCR.

[0377] Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR: 5′-gtcccccacagatgcagaca aagaacttgg agattgggtc atcacaatgt caccagtgga-3′; (HKSPR11; SEQID No. 49) 5′-ccaagttctt tgtctgcatc agtaggagac agggtcacca tcacctgc-3′;(HKCDF11; SEQ ID No. 50) 5′-agtgtgccgg gtggatgccc agtaaatcag tagtttaggagctttccctg gtttctg-3′; (HKCDR12; SEQ ID No. 51) 5′-tgggcatcca cccggcacactggggtccca agcaggttta gtggcagt-3′; (HKCDF22; SEQ ID No. 52)5′-ataactacta tattgctgac agtaataggt tgcaaaatcc tccggctgca gactagagatggt-3′; (HKCDR22; SEQ ID No. 53) and 5′-cagcaatata gcagctatcg gacgttcggtcaaggcacca aggtggaaat caaacggact gtg-3′. (HKCF12; SEQ ID No. 54)

[0378] 2) Construction of Plasmid pCR3.1/M2-1 (Cloning of HumanizedTRA-8 Light Chain)

[0379] LM2-DNA fragment as defined in SEQ ID No. 55 of the SequenceListing coding for the amino acid sequence as defined in SEQ ID No. 46of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

[0380] a) First step PCR

[0381] LM2-F 1-DNA fragment coding for a secretion signal sequence and aportion of FRL₁ region with a Hind III restriction enzyme cleavage siteadded at the 5′-end is prepared under the following conditions. Thetemplate plasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0909816 A1.

[0382] Composition of the reaction solution:

[0383] plasmid pHSGHM17 DNA (European patent application EP 0 909 816A1), 25 ng

[0384] oligonucleotide primer 7AL1P, 50 pmol

[0385] oligonucleotide primer HKSPR11, 50 pmol

[0386] dNTPs cocktail, 5 μl

[0387] 10×PCR buffer, 5 μl

[0388] ampliTaq DNA polymerase (PerkinElmer), 2.5 units

[0389] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0390] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0391] LM2-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂,and CDRL₂ and is prepared under the following conditions.

[0392] Composition of the reaction solution:

[0393] plasmid pL28 DNA, 25 ng

[0394] oligonucleotide primer HKCDF11, 50 pmol

[0395] oligonucleotide primer HKCDR12, 50 pmol

[0396] dNTPs cocktail, 5 μl

[0397] 10×PCR buffer, 5 μl

[0398] ampliTaq DNA polymerase, 2.5 units

[0399] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0400] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0401] LM2-F3-DNA fragment coding for CDRL₂, FRL₃, and a portion ofCDRL₃ is prepared under the following conditions.

[0402] Composition of the reaction solution:

[0403] plasmid pSRPDHH DNA (European patent application EP 0 909 816A1), 25 ng

[0404] oligonucleotide primer HKCDF22, 50 pmol

[0405] oligonucleotide primer HKCDR22, 50 pmol

[0406] dNTPs cocktail, 5 μl

[0407] 10×PCR buffer, 5 μl;

[0408] ampliTaq DNA polymerase, 2.5 units

[0409] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0410] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0411] LM2-F4-DNA fragment coding for CDRL₃, FRL₄ and the constantregion with an EcoRI restriction enzyme cleavage site added at the3′-end is prepared under the following conditions.

[0412] Composition of the reaction solution:

[0413] plasmid pSRPDHH DNA, 25 ng

[0414] oligonucleotide primer HKCF12, 50 pmol

[0415] oligonucleotide primer 7ALCN, 50 pmol

[0416] dNTPs cocktail, 5 μl

[0417] 10×PCR buffer, 5 μl

[0418] ampliTaq DNA polymerase, 2.5 units

[0419] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0420] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0421] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor

[0422] b) Second Step PCR

[0423] LM2-DNA in which above described LM2-F1-DNA, LM2-F2-DNA,LM2-F3-DNA and LM2-F4-DNA fragments are fused is prepared under thefollowing conditions.

[0424] Composition of the reaction solution:

[0425] Gel fragment of LM2-F 1-DNA prepared in the first step PCR,

[0426] Gel fragment of LM2-F2-DNA prepared in the first step PCR,

[0427] Gel fragment of LM2-F3-DNA prepared in the first step PCR,

[0428] Gel fragment of LM2-F4-DNA prepared in the first step PCR

[0429] oligonucleotide primer 7AL1P, 50 pmol

[0430] oligonucleotide primer 7ALCN, 50 pmol

[0431] dNTPs cocktail, 5.0 μl

[0432] 10×PCR buffer, 5.0 μl

[0433] ampliTaq DNA polymerase, 2.5 units

[0434] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0435] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0436] The thus prepared LM2-DNA fragment is inserted into plasmidpCR3.1DNA using Eukaryotic TA cloning Kit (Invitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 are confirmed by the dideoxymethod (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin Elmer AppliedBiosystems, Japan).

[0437] The resulting plasmids are designated pCR3.1/M2-1 (the plasmidcarrying cDNA encoding the light chain variable region of humanizedTRA-8 and a human Ig light chain constant region).

[0438] The obtained plasmid pCR3.1/M2-1 containing LM2-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

[0439] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM2-DNA fragment,that have been digested with the restriction enzymes Hind III and EcoRI, are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co.Ltd.). Then, E. coli DH5α is transformed with the ligated DNA and spreadonto LB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM2-DNA fragment is selected by 1%agarose gel electrophoresis.

[0440] As a result of the above procedure, plasmid pHSG/M2-1-4 carryinga fusion fragment of the variable region of the humanized LM2 TRA-8light chain and the constant region of human IgK chain is obtained. Thetransformant E coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M2-1-4 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7563.

[0441] 3) Construction of Plasmid pSR/M2-1 (Expression Plasmid forHumanized LM2 TRA-8 Light Chain)

[0442] The obtained plasmid pHSG/M2-1-4 carrying a fusion fragment ofthe variable region of the humanized LM2 TRA-8 light chain and theconstant region of human IgK chain is digested with the restrictionenzymes Hind III and EcoR I.

[0443] One μg of cloning plasmid pSRPDHH DNA (European patentapplication EP 0-909-816-A1) is digested with the restriction enzymesHind III and EcoR I, and then dephosphorylated with CIP. The resultingdephosphorylated pSRPDHH DNA and HindIII-EcoRI DNA fragment obtainedfrom pHSG/M2-1-4 are ligated using DNA Ligation Kit Version 2.0 (TakaraSyuzo, Co. Ltd.). Then, E. coli DH5α is transformed with the ligated DNAand spread onto LB agar. The transformants obtained are cultured inliquid LB medium containing 100 μg/ml ampicillin, and plasmid DNA isextracted from the resulting culture according to the alkaline-SDSmethod. The insertion and orientation of the desired DNA fragment inpSRPDHH vector is confirmed by DNA sequencing using a gene sequenceanalyzer (ABI Prism 3700 DNA Analyzer; Applied Biosystems).

[0444] The resulting expression plasmid carrying cDNA encoding the lightchain of humanized TRA-8 is designated pSR/M2-1.

EXAMPLE 20 Production of Humanized Antibody

[0445] Transfection of COS-7 cells, i.e., a cell line derived from amonkey kidney, with the expression plasmids for the humanized TRA-8heavy chain and the humanized TRA-8 light chain obtained above isconducted by FUGENE6 transfection reagent methods (Boehringer MannheimBiochemica) according to the instruction manual provided with the kit.

[0446] COS-7 cells (American Type Culture Collection No. CRL-1651) aregrown to semi-confluent (3×10⁶ cells/dish) in a culture dish (culturearea: 57 cm²; Sumitomo Bakelite) containing Dulbecco's Modified Eaglemedium (hereinafter referred to as “D-MED”; Gibco BRL) supplemented with10% fetal calf serum (hereinafter abbreviated as “FCS”; Moregate).

[0447] In the meantime, 10 μg/dish (total 5 dishes) of the humanized DR5heavy chain expression plasmid DNA (pHA15-1) and 10 μg/dish of thehumanized DR5 light chain expression plasmid DNA prepared by thealkaline-SDS method and cesium chloride density gradient centrifugationare mixed, and then precipitated with ethanol, followed by suspending in5 μl/dish of dH₂O.

[0448] After 15 μl/dish of FUGENE6 Transfection regent is mixed with 180μl/dish D-MEM without FCS, this FUGENE solution (185 μl/dish) is mixedwith 5 μl/dish DNA solution containing 10 μg/dish of the humanized DR5heavy chain expression plasmid DNA and 10 μg/dish of the humanized DR5light chain expression plasmid DNA. After 15 minutes incubation at roomtemperature, the obtained plasmid suspension (200 μl) is added to thepreviously prepared COS-7 plates. After incubating in 5% CO₂ at 37° C.for 24 hours, the culture medium is changed with D-MEM without FCS.After incubating in 5% CO₂ at 37° C. for 72 hours, the culturesupernatant is recovered to purify the expression products in thesupernatant fluids. By the method as described above, COS-7 cells aretransfected with each of the following plasmid combinations:

[0449] (A): no plasmid DNA

[0450] (B): cotransfection of pHB 14-1 and pSR/M2-1

[0451] The culture is then centrifuged (1,000 r.p.m., 5 minutes) andcollected the supernatant. The supernatant is centrifuged again (9,800r.p.m., 15 minutes) and filtrated with 0.45 μm filter (ADVANTEC TOYODISMIC-25cs, Cat # 25CS045 AS). The purification of IgG from thefiltrates are achieved using Protein G-POROS affinity chromatography(Applied Biosystems) under the following conditions:

[0452] HPLC: BioCAD 700E (Applied Biosystems)

[0453] column: ProteinG-ID sensor cartridge (column size: 2.1 mmID×30 mmLD, bed volume: 0.1 ml; Cat # 2-1002-00, Applied Biosystems)

[0454] elution buffer: 0.1M Glycine-HCl (pH 2.5)

[0455] neutralization buffer: 1M Tris-HCl (pH 8.5)

[0456] detection: 280 nm

[0457] flow rate: 1 ml/min

[0458] fraction size: 0.5 ml/0.5 min

[0459] fraction tube: 1.5 ml polypropylene microtube

[0460] temperature: 4° C.

[0461] After all the filtrates are applied to column, 30 ml of PBS(Sigma, Cat # 1000-3) is used to wash column. When the elution buffer isapplied, fraction collector started. Each fraction microtube previouslycontained 55 μl of 1M NaCl, 110 μl of neutralization buffer and 74 μl of2 mg/ml bovine serum albumin (Sigma, Cat # A-7030) in PBS. The fractionsfrom No. 8 through No. 10 are collected and dialyzed against 1 liter PBS(pH 7.5) at 4° C. for 1 day using Slide-A lyzer (Pierce, Cat # 66450).The dialysis buffer is changed twice.

[0462] Verification of the expression of the humanized antibodies andquantitative assay of the expression products in the culture supernatantfluids prepared is performed by ELISA with an antibody againstanti-human IgG.

[0463] To each well of a 96-well plate (MaxiSorp, Nunc), 100 μl of goatanti-human IgG Fc specific polyclonal antibody (Kappel) dissolved at thefinal concentration of 0.5 μg/ml in adsorption buffer (0.05 M sodiumhydrogencarbonate, 0.02% sodium azide, pH 9.6) is added and the plate isincubated at 37° C. for 2 hours to cause adsorption of the antibody.Then, the plate is washed with 350 μl of PBS(−) containing 0.05%Tween-20 (BioRad) (hereinafter referred to as “PBS-T”) five times. Tothe wells after washing, the culture supernatant diluted with D-MEMcontaining 10% FCS is added and incubated at 37° C. for 2 hours. Afterwashing again with PBS-T, 100 μl of alkaline phosphatase-labeled goatanti-human IgG Fc specific polyclonal antibody (Jackson Immuno ResearchLab.) diluted 10,000-fold with PBS-T is added to each well and incubatedat 37° C. for 2 hours. After washing again with PBS-T, a substratesolution of p-nitrophenyl phosphate obtained from Alkaline PhosphataseSubstrate kit (Bio Rad) is added according to the instruction manualprovided with the kit. After incubating at 37° C. for 0.5 to 1 hour, theabsorbance at 405 nm is measured. In the present experiments, humanplasma immunoglobulin G subclass 1 (IgG1) (Biopure AG) diluted withD-MEM containing 10% FCS to certain concentrations is used asconcentration reference samples of the humanized DR5 antibodiescontained in the culture supernatant fluids.

[0464] As a result, the expression and purified products in the culturesupernatant are detected specifically with the anti-human IgG antibody.The amount of human IgG antibody is 8.96 μg (800 μl).

EXAMPLE 21 Apoptosis-Inducing Activity of Humanized Antibody

[0465] Jurkat cells (ATCC No. TIB-152), are used to examine theapoptosis-inducing activity of the purified humanized TRA-8 antibody.

[0466] Jurkat cells cultured in RPM11640 medium with 10% FCS (Gibco BRL)at 37° C. for 3 days in the presence of 5% CO₂ are dispensed into eachwell of a 96-well microplate (Sumitomo Bakelite) at 50 μl per well. Thehumanized TRA-8 prepared in Example 20 are adjusted to have theconcentration of the final product of interest of 100 ng/ml withRPMI1640 medium containing 10% FCS by estimating their concentrations inthe fluids according to the method described in Example 20. Each of thesolutions of the expression products thus adjusted to 100 ng/ml is usedto produce serial dilutions by repeating serial 2-fold dilution withRPM1640 containing 10% FCS. Each of the diluted humanized TRA-8 solutionis added to each well at 50 μl per well. After reacting at 37° C. for 12hours, 50 μl of 25 μM PMS (phenazine methosulfate; Sigma Chemical Co.)containing 1 mg/ml XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanirideinner salt; Sigma Chemical Co.) is added (final concentrations of 250μg/ml for XTT and 5 μM for PMS). After incubating for 3 hours, theabsorbance at 450 nm of each well is measured to calculate the cellviability by using the reduction ability of mitochondria as the index.

[0467] The viability of the cells in each well is calculated accordingto the following formula:

Viability (%)=100×(a−b)/(c−b)

[0468] wherein “a” is the measurement of a test well, “b” is themeasurement of a well with no cells, and “c” is the measurement of awell with no antibody added.

[0469] As a result, the expression product prepared in Example 20(humanized TRA-8) is demonstrated to induce apoptosis in cells of Tlymphoma cell line expressing human DR5 antigen.

EXAMPLE 22 Reactivity of TRA-8 to Various DR5 Molecules

[0470] In order to determine the reactivity of TRA-8 to various DR5molecules, the reactivity of TRA-8 is examined using activatedlymphocytes as follows.

[0471] First, peripheral blood samples are taken from a human (30 ml),marmoset (3 ml), and cynomolgus monkey (20 ml). The blood samples had 1ml of heparin (Novoheparin; Novo) added to them and the samples are thenslowly layered over an equal volume of Ficoll-Paque PLUS solution((Amersham Pharmacia Biotech.) specific gravity: 1.077 for all exceptcynomolgus monkey, which had a specific gravity of 1.072) andcentrifuged at 1,700 r.p.m. for 30 minutes in order to obtain a fractionof peripheral blood mononuclear cells. This mononuclear cell fraction iswashed twice with Hanks' balanced salt solution and then suspended inRPMI 1640 medium with 10% v/v FCS to a cell density of 1×10⁶ cells/ml.Phytohemagglutinin-P (PHA-P, Sigma Chemicals, Co.) is added to theresulting suspension to a final concentration of 5 μg/ml and the sampleincubated at 37° C. under 5% v/v CO₂ for 24 hours. After this time, thecells are recovered by centrifugation, washed and resuspended in RPMI1640 medium containing 10% v/v FCS. Then, to activate the recoveredcells, interleukin-2 (Amersham Pharmacia Biotech.) is added to thesuspension to a final concentration of 10 units/ml, and this isincubated at 37° C. under 5% v/v CO₂ for 72 hours.

[0472] An amount of the activated preparation calculated to contain1×10⁶ activated lymphocyte cells is placed in a test tube and eithersuspended in 50 μl of 0.5, 1, 5, 10 μg/ml of TRA-8 in PBS or 50 μl ofPBS alone. The resulting suspension is allowed to stand on ice for 1hour, after which the cells are washed 3 times with aliquots of 500 μlof PBS and then suspended in 50 μl of 20 μg/ml FITC-labeled anti-mouseIgG antibody (Bioresource) in PBS. Using the cells suspended in 500 μlof PBS as controls, the fluorescence intensities are measured, using aflow cytometer (FACSCalibur; Becton Dickinson).

[0473] Distributions of cell numbers by fluorescence intensity areobtained and the proportions of the numbers of the stained cells tothose of total cells are calculated. Further, each Kd value iscalculated using the concentration of TRA-8 and the proportions of thenumbers of the stained cells to those of total cells. Each frequency ofreactivity to activated lymphocytes of human, marmoset, and cynomologusmonkey is almost same. Accordingly, TRA-8 is able to bind a wide rangeof primate DR5 including human against which TRA-8 is originallyprepared.

EXAMPLE 23 Escalating Dose Study of TRA-8 in Marmosets

[0474] An escalating dose preliminary toxicity study of TRA-8 isperformed using I male and 1 female marmoset. Three sets of singleintravenous dosing, which are separated by a 7-day withdrawal period,are carried out. The dose of TRA-8 is set at 50, 250 and 1250 μg/body.Forty-eight hours after each treatment, blood is collected from thefemoral vein and the plasma is prepared. Plasma aspartateaminotransferase and alanine aminotransferase activities are measuredusing an analyzer (FUJI DRI-CHEM: Fuji Film Medical Co., Ltd.). Allblood is taken without any anesthetization. As a result, no evidencesindicating hepatic injury are noted in plasma biochemical examinationafter each treatment.

EXAMPLE 24 In vitro and in vivo Pharmacological Studies of TRA-8 AgainstCancer Cells

[0475] In order to determine whether TRA-8 has the therapeutic efficacyin anti-cancer therapy, in vitro killing activity of TRA-8 using variouscancer cell lines is examined as follows.

[0476] Various cancer cells (2-8×10³ cells/50 μl) cultured in RPM11640medium (for Jurkat), DMEM medium (for HCT-116), MEM-R (for WiDr), orDMEM-F12 (for COL2-Jck) obtained from Gibco BRL with 10% FCS (Gibco BRL)at 37° C. in the presence of 5% CO₂ are dispensed into each well of a96-well microplate (Sumitomo Bakelite). TRA-8 are adjusted to have theconcentration of the final product of interest of 100 ng/ml with mediumcontaining 10% FCS. The TRA-8 solution (100 ng/ml) is used to produceserial dilutions by repeating serial 2-fold dilution with mediumcontaining 10% FCS. Each of the diluted TRA-8 solution is added to eachwell at 50 μl per well and incubated at 37° C. After reacting at 37° C.for 72 hours, 50 μl of 25 μM PMS (phenazine methosulfate; Sigma ChemicalCo.) containing 1 mg/ml XTT is added (final concentrations of 250 μg/mlfor XTT and 5 μM for PMS). After incubating for 3 hours, the absorbanceat 450 nm of each well is measured to calculate the cell viability byusing the reduction ability of mitochondria as the index.

[0477] The viability of the cells in each well is calculated accordingto the following formula:

Viability (%)=100×(a−b)/(c−b)

[0478] wherein “a” is the measurement of a test well, “b” is themeasurement of a well with no cells, and “c” is the measurement of awell with no antibody added.

[0479] The results are shown in Table 3, below. TABLE 3 ED50 Cells(μg/ml) Jurkat 0.001-0.01 HCT-116 0.004-0.02 WiDr 0.007-0.03 COL2-Jck2.28

[0480] Various cancer cell lines are strongly induced apoptosis by TRA-8under the in vitro conditions.

[0481] Furthermore, the in vivo anti-tumor effect of TRA-8 in nude micetransplanted with WiDr cells is determined, because TRA-8 is notcross-reactive with murine DR5.

[0482] TRA-8 anti-human DR5 antibody is administered to nude micebearing human xenografts that express the human DR5 molecule. The miceused were 6 week-old BALb/c nude/nude mice (female, from Clea JapanInc.), which were transplanted with human colon cancer cell lines WiDr(5 mm³). At one day after tumor transplantation, these transplanted miceare daily treated with the intra-articular injection of TRA-8 (5μg/body) to 14 times. WiDr tumor growth is daily determined by the sizeof the tumor mass. The results are shown in Table 4, below. TABLE 4 8days 11 days 15 days 18 days 22 days 25 days Control (PBS) 196 ± 55 249± 77 469 ± 149 584 ± 230 833 ± 274 1193 ± 419 SD TRA-8 158 ± 78  97 ± 30155 ± 60  195 ± 58  365 ± 91   530 ± 135 SD

[0483] In this model, while all untreated animals exhibited visibletumor growth, tumor growth in TRA-8 treated animals is inhibited asdemonstrated by the size of tumor. This result indicated that TRA-8 iseffective in the elimination of tumor cells in vivo.

EXAMPLE 25 Combination Study of TRA-8

[0484] Human prostate cancer cell line PC-3 is obtained from AmericanTissue Culture Collection (ATCC) and maintained in F-12K NutrientMixture (21127-022, Gibco BRL) containing 10% fetal bovine serum (FBS,Hyclone), 1% L-Glutamine-200 mM (25030-149, Gibco BRL) and 0.5%Penicillin Streptomycin Solution (P-7539, Sigma). RPMI1640 medium(MED-008, IWAKI) supplemented with 10% FBS and 0.5% PenicillinStreptomycin Solution is used in the following experiment. Exponentiallygrowing PC-3 cells are collected by trypsinization and washed twice withfresh medium. The cells are then counted, resuspended in fresh medium ata density of 5×10⁴ cells/ml and distributed in triplicate intoflat-bottomed 96 well plates (3598, Coming-Coster) in a total volume of100 μl/well one day before the start of the experiment. A representativeanti-cancer drug, Paclitaxel (169-18611, Wako) dissolved indimethylsulfoxide (10 mg/ml) is diluted in fresh medium and then addedto the 96-well plates containing the cells at 50 μl/well. The finalconcentrations of dimethylsulfoxide are less than 0.1%. After incubationfor 24 hr at 37° C. in 5% CO₂ atmosphere, TRA-8 diluted in fresh mediumis added to the wells. After incubation for a further 24 hr, 50 μl ofMinimum Essential Medium (11095-098, Gibco BRL) containing 1 mg/ml ofXTT and 25 mM of PMS is added to the wells and the plates are incubatedfor 6 hr. OD450 is then measured by SPECTRA MAX 250 (Molecular Devices)and the cell viability is calculated as follows.

Cell viability (%)=(OD450 for the well containing cells treated withTaxol and/or TRA-8 (agent(s))−OD450 for the well containing neithercells nor agent)×100/(OD450 for the well containing cells with noagent−OD450 for the well containing neither cells nor agent)

[0485] The result of the above assay for TRA-8 combined with arepresentative anti-cancer drug, Paclitaxel, is followed. Paclitaxelreduced the cell viability of PC-3 cells but more than 40% of thesignals indicating viable cancer cells still remained at concentrationsof up to 200 nM. Notably, the addition of 0.1 ng/ml of TRA-8 greatlydecreased the cell viability of the cancer cells, up to 10%, even thoughno reduction in cell viability is seen after a single application ofTRA-8 at this concentration. This result clearly indicates that TRA-8exhibited anti-cancer activity synergistically when combined with otheranti-cancer drugs.

EXAMPLE 26 Analysis of Other Type Humanized Antibodies of TRA-8

[0486] (1) Designing Humanized Antibodies

[0487] Construction of a humanized version of TRA-8 is performed by themethod generally known as CDR grafting. mAB58′CL antibody is used as anacceptor as described in Reference Example 2 and the CDR regions ofTRA-8 antibody is grafted on the acceptor. In the framework region, someamino acids are grafted on the acceptor from either TRA-8 or humanconsensus sequences by the criteria given by Queen et al. (Proc. Natl.Acad. Sci. USA 86, 10029-10033, (1989)) and humanized TRA-8 sequencesare constructed as described hereinbelow.

[0488] (2) Construction of Plasmid Carrying the Heavy Chain VariableRegion DNA of Other Types Humanized or Mouse TRA-8

[0489] As shown in SEQ ID No. 56 of the Sequence Listing, Hitype-humanization of the amino acid sequences of the heavy chain of themouse anti-human DR5 antibody TRA-8 entailed replacing the 3rd aminoacid (methionine), the 13th amino acid (lysine), the 19th amino acid(lysine), the 40th amino acid (threonine), the 42nd amino acid (glutamicacid), the 44th amino acid (arginine), the 84th amino acid (serine), the88th amino acid (serine), the 93rd amino acid (methionine), the 114thamino acid (threonine), the 115th amino acid (leucine) with glutamine,glutamine, arginine, alanine, glycine, glycine, asparagine, alanine,valine, leucine, and valine, respectively.

[0490] As shown in SEQ ID No. 59 of the Sequence Listing, H3type-humanization of the amino acid sequences of the heavy chain of themouse anti-human DR5 antibody TRA-8 entailed replacing the 13th aminoacid (lysine), the 19th amino acid (lysine), the 40th amino acid(threonine), the 42nd amino acid (glutamic acid), the 44th amino acid(arginine), the 88th amino acid (serine), the 93rd amino acid(methionine), the 114th amino acid (threonine), the 115th amino acid(leucine) with glutamine, arginine, alanine, glycine, glycine, alanine,valine, leucine, and valine, respectively.

[0491] As shown in SEQ ID No. 60 of the Sequence Listing, H4type-humanization of the amino acid sequences of the heavy chain of themouse anti-human DR5 antibody TRA-8 entailed replacing the 13th aminoacid (lysine), the 19th amino acid (lysine), the 88th amino acid(serine), the 93rd amino acid (methionine), the 114th amino acid(threonine), the 115th amino acid (leucine) with glutamine, arginine,alanine, valine, leucine, and valine, respectively.

[0492] As shown in SEQ ID No. 61 of the Sequence Listing, the plasmidcarrying the heavy chain variable region DNA of chimeric TRA-8 isdesignated as “M type”. In addition, humanized TRA-8 described inExample 17 and 18 is designated as “H2 type”.

[0493] The plasmids carrying DNA encoding heavy chain variable region ofhumanized or chimeric TRA-8 are constructed as follows.

[0494] PCR is used to construct the following DNA sequences, each ofwhich comprised described above:

[0495] The following 24 oligonucleotide are synthesized: 5′-ttggataagcttggcttgac ctcaccatgg gatggagctg tatcatcctc ttcttggtag caacagctac (A;SEQ ID No. 32) aggtgtccac-3′; 5′-tctgaagtaa tgctggtgga gtctgggggaggcttagtac agcctggagg gtccctgaga (B; SEQ ID No. 33) ctctcctgtgcagcctctgg-3′; 5′-tctgaagtac agctggtgga gtctggggga ggcttagtac agcctggagggtccctgaga (B2; SEQ ID No. 57) ctctcctgtg cagcctctgg-3′; 5′-tctgaagtaatgctggtgga gtctggggga ggcttagtaa agcctggagg gtccctgaaa (B3; SEQ ID No.66) ctctcctgtg cagcctctgg-3′; 5′-attcactttc agtagttatg taatgtcttgggttcggcag gcaccaggga agggtctgga (C; SEQ ID No. 34) gtgggttgcaaccattagta-3′; 5′-attcactttc agtagttatg taatgtcttg ggttcggcag actccagagaagaggctgga gtgggttgca (C2; SEQ ID No. 64) accattagta-3′; 5′-gtggtggtagttacacctac tatccagaca gtgtgaaggg ccgattcacc atctccagag (D; SEQ ID No.35) acaatgccaa gaacaccctg-3′; 5′-tatctgcaaa tgaacagtct gagagcagaggacacggctg tttattactg tgcaagaagg (E; SEQ ID No. 36) ggtgactctatgattacgac-3′; 5′-tatctgcaaa tgagcagtct gagagcagag gacacggctg tttattactgtgcaagaagg (E2; SEQ ID No. 62) ggtgactcta tgattacgac-3′; 5′-tatctgcaaatgagcagtct gagatctgag gacacggcta tgtattactg tgcaagaagg (E3; SEQ ID No.67) ggtgactcta tgattacgac-3′; 5′-ggactactgg ggccaaggga ccctggtcacagtctcctca gcctccacc aagggcccat cggtc- (F; SEQ ID No. 37) 3′;5′-ggactactgg ggccaaggga ccactctcac agtctcctca gcctccacc aagggcccatcggtc- (F2; SEQ ID No. 68) 3′; 5′-ctaccaagaa gaggatgata cagctccatcccatggtgag gtcaagccaa gcttatccaa-3′; (G; SEQ ID No. 38) 5′-tctcagggaccctccaggct gtactaagcc tcccccagac tccaccagca ttacttcaga (H; SEQ ID No.39) gtggacacct gtagctgttg-3′; 5′-tctcagggac cctccaggct gtactaagcctcccccagac tccaccagct gtacttcaga (H2; SEQ ID No. 58) gtggacacctgtagctgttg-3′; 5′-tttcagggac cctccaggct ttactaagcc tcccccagac tccaccagcattacttcaga (H3; SEQ ID No. 69) gtggacacct gtagctgttg-3′; 5′-tccagacccttccctggtgc ctgccgaacc caagacatta cataactact gaaagtgaat (I; SEQ ID No.40) ccagaggctg cacaggagag-3′; 5′-tccagcctct tctctggagt ctgccgaacccaagacatta cataactact gaaagtgaat (I2; SEQ ID No. 65) ccagaggctgcacaggagag-3′; 5′-ctctggagat ggtgaatcgg cccttcacac tgtctggata gtaggtgtaactaccaccac tactaatggt (J; SEQ ID No. 41) tgcaacccac-3′; 5′-ccttcttgcacagtaataaa cagccgtgtc ctctgctctc agactgttca tttgcagata cagggtgttc (K;SEQ ID No. 42) ttggcattgt-3′; 5′-ccttcttgca cagtaataaa cagccgtgtcctctgctctc agactgttca tttgcagata cagggtgttc (K2; SEQ ID No. 63)ttggcattgt-3′; 5′-ccttcttgca cagtaataca tagccgtgtc ctcagatctc agactgctcatttgcagata cagggtgttc (K3; SEQ ID No. 70) ttggcattgt-3′; 5′-gaccgatgggcccttggtgg aggctgagga gactgtgacc agggtccctt ggccccagta (L; SEQ ID No.43) gtccgtcgta atcatagagt cacc-3′ and 5′-gaccgatggg cccttggtggaggctgagga gactgtgaga gtggtccctt ggccccagta (L2; SEQ ID No. 71)gtccgtcgta atcatagagt cacc-3′. The following 2 PCR primers aresynthesized as described above: 5′-ttggataagc ttggcttgac-3′; and (P1;SEQ ID No. 44) 5′-gaccgatggg cccttggtgg a-3′. (P2; SEQ ID No. 45)

[0496] The synthesis of H1 type DNA encoding a polypeptide chaincomprising a secretion signal sequence, a variable region of humanizedTRA-8 heavy chain and the 8 amino acid residues at the N-terminus of theIgG-CH1 region is performed using a combination of PCR respectively.

[0497] The H1 type-DNA fragment is prepared as follows.

[0498] Composition of the PCR reaction solution:

[0499] oligonucleotide A, 10 pmol;

[0500] oligonucleotide B2, 10 pmol;

[0501] oligonucleotide C, 10 pmol;

[0502] oligonucleotide D, 10 pmol;

[0503] oligonucleotide E, 10 pmol;

[0504] oligonucleotide F, 10 pmol;

[0505] oligonucleotide G, 10 pmol;

[0506] oligonucleotide H2, 10 pmol;

[0507] oligonucleotide I, 10 pmol;

[0508] oligonucleotide J, 10 pmol;

[0509] oligonucleotide K, 10 pmol;

[0510] oligonucleotide L, 10 pmol;

[0511] oligonucleotide primer P1, 2 μM;

[0512] oligonucleotide primer P2, 2 μM;

[0513] 10×Pyrobest buffer II, 10 μl;

[0514] dNTP mix, 8 μl;

[0515] Pyrobest DNA polymerase, 0.5 μl; and

[0516] Redistilled water to a final volume of 50 μl.

[0517] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 5 minutes, after which a cycle of heating to 98° C.for 10 second, 55° C. for 30 second and 72° C. for 1 minute, is repeated7 times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

[0518] After phenol extraction and ethanol precipitation, the resultingDNA precipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to H1 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean Spin Kit (BIO 101, CA, USA). After phenolextraction, the eluted DNA is then concentrated by centrifugation at7,500×g, followed by ethanol precipitation, and finally dissolved in 5μl of distilled water.

[0519] The synthesis of H3 type DNA encoding a polypeptide chaincomprising a secretion signal sequence, a variable region of humanizedTRA-8 heavy chain and the 8 amino acid residues at the N-terminus of theIgG-CH1 region is performed using a combination of PCR respectively.

[0520] The H3 type-DNA fragment is prepared as follows.

[0521] Composition of the PCR reaction solution:

[0522] oligonucleotide A, 10 pmol;

[0523] oligonucleotide B, 10 pmol;

[0524] oligonucleotide C, 10 pmol;

[0525] oligonucleotide D, 10 pmol;

[0526] oligonucleotide E2, 10 pmol;

[0527] oligonucleotide F, 10 pmol;

[0528] oligonucleotide G, 10 pmol;

[0529] oligonucleotide H, 10 pmol;

[0530] oligonucleotide I, 10 pmol;

[0531] oligonucleotide J, 10 pmol;

[0532] oligonucleotide K2, 10 pmol;

[0533] oligonucleotide L, 10 pmol;

[0534] oligonucleotide primer P1, 2 μM;

[0535] oligonucleotide primer P2, 2 μM;

[0536] 10×Pyrobest buffer II, 10 μl;

[0537] dNTP mix, 8 μl;

[0538] Pyrobest DNA polymerase, 0.5 μl; and

[0539] Redistilled water to a final volume of 50 μl.

[0540] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 5 minutes, after which a cycle of heating to 98° C.for 10 second, 55° C. for 30 second and 72° C. for 1 minute, is repeated7 times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

[0541] After phenol extraction and ethanol precipitation, the resultingDNA precipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to H3 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

[0542] The synthesis of H4 type DNA encoding a polypeptide chaincomprising a secretion signal sequence, a variable region of humanizedTRA-8 heavy chain and the 8 amino acid residues at the N-terminus of theIgG-CH1 region is performed using a combination of PCR respectively.

[0543] The H4 type-DNA fragment is prepared as follows.

[0544] Composition of the PCR reaction solution:

[0545] oligonucleotide A, 10 pmol;

[0546] oligonucleotide B, 10 pmol;

[0547] oligonucleotide C2, 10 pmol;

[0548] oligonucleotide D, 10 pmol;

[0549] oligonucleotide E2, 10 pmol;

[0550] oligonucleotide F, 10 pmol;

[0551] oligonucleotide G, 10 pmol;

[0552] oligonucleotide H, 10 pmol;

[0553] oligonucleotide I2, 10 pmol;

[0554] oligonucleotide J, 10 pmol;

[0555] oligonucleotide K2, 10 pmol;

[0556] oligonucleotide L, 10 pmol;

[0557] oligonucleotide primer PI, 2 μM;

[0558] oligonucleotide primer P2, 2 μM;

[0559] 10×Pyrobest buffer II, 10 μl;

[0560] dNTP mix, 8 μl;

[0561] Pyrobest DNA polymerase, 0.5 μl; and

[0562] Redistilled water to a final volume of 50 μl.

[0563] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 5 minutes, after which a cycle of heating to 98° C.for 10 second, 55° C. for 30 second and 72° C. for 1 minute, is repeated7 times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

[0564] After phenol extraction and ethanol precipitation, the resultingDNA precipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under WV light. The DNA bandscorresponding to H4 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

[0565] The synthesis of M type DNA encoding a polypeptide chaincomprising a secretion signal sequence, a variable region of chimericTRA-8 heavy chain and the 8 amino acid residues at the N-terminus of theIgG-CH1 region is performed using a combination of PCR respectively.

[0566] The M type-DNA fragment is prepared as follows.

[0567] Composition of the PCR reaction solution:

[0568] oligonucleotide A, 10 pmol;

[0569] oligonucleotide B3, 10 pmol;

[0570] oligonucleotide C2, 10 pmol;

[0571] oligonucleotide D, 10 pmol;

[0572] oligonucleotide E3, 10 pmol;

[0573] oligonucleotide F2, 10 pmol;

[0574] oligonucleotide G, 10 pmol;

[0575] oligonucleotide H3, 10 pmol;

[0576] oligonucleotide I2, 10 pmol;

[0577] oligonucleotide J, 10 pmol;

[0578] oligonucleotide K3, 10 pmol;

[0579] oligonucleotide L2, 10 pmol;

[0580] oligonucleotide primer P1, 2 μM;

[0581] oligonucleotide primer P2, 2 μM;

[0582] 10×Pyrobest buffer II, 10 μμl;

[0583] dNTP mix, 8 μl;

[0584] Pyrobest DNA polymerase, 0.5 μl; and

[0585] Redistilled water to a final volume of 50 μl.

[0586] The PCR reaction is conducted as follows. The solution is firstheated at 94° C. for 5 minutes, after which a cycle of heating to 98° C.for 10 second, 55° C. for 30 second and 72° C. for 1 minute, is repeated7 times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

[0587] After phenol extraction and ethanol precipitation, the resultingDNA precipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to M type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

[0588] The resulting, each extracted DNA (HI type, H3 type, H4 type, andM type) is cloned using pGEM-T Easy vector (Promega) as follows:

[0589] The DNA fragment recovered from the PCR reaction (H1, H3, H4 orM), 5 μl;

[0590] 10×Taq polymerase buffer, 1 μl;

[0591] dNTP mixture, 1 μl

[0592] Taq polymerase (5 unit/ml), 1 μl; and

[0593] redistilled water to a final volume of 10 μl.

[0594] After the above each solution is reacted at 70° C. for 30minutes, each DNA solution and pGEM-T Easy vector are ligated using aDNA Ligation Kit Version 2.0 (Takara Shuzo Co., Ltd.) using themanufacturer's protocol.

[0595] After 4 hours incubation at 15° C., 2 μl of the incubatedreaction solution is mixed with 100 μl of competent E. Coli strain JM109at a cell density of 1-2×10⁹ cells/ml (Takara Shuzo Co., Ltd.), and themixture is kept on ice for 30 minutes, then at 42° C. for 30 seconds,and again on ice for 1 minute. Then, 500 μl of SOC medium (2% v/vtryptone, 0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM w/vpotassium chloride, 1 mM magnesium chloride, and 20 mM glucose) is addedthe mixture, which is incubated for a further hour, with shaking.Transformant strains are then isolated, and plasmid DNA is prepared fromthe strains as described in “Molecular Cloning: A Laboratory Manual”.The nucleotide sequence of this DNA encoding the heavy chain ofhumanized or mouse TRA-8 is confirmed by the dideoxy method,respectively (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin Elmer AppliedBiosystems, Japan).

[0596] The resulting plasmid is designated pHA15 (the plasmid carryingcDNA encoding the HI-type heavy chain of humanized TRA-8), pHC10 (theplasmid carrying cDNA encoding the H3-type heavy chain of humanizedTRA-8), pHD21 (the plasmid carrying cDNA encoding the H4-type heavychain of humanized TRA-8), and pM11 (the plasmid carrying cDNA encodingthe heavy chain of chimeric TRA-8). The transformant E coli strainsharboring these plasmid, designated as E. coli JM109/pHA15, E. coliJM109/pHC10, E. coli JM109/pHD21, and E. coli JM109/pM11 were depositedwith International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, 1-1, Higashi 1 chomeTsukuba-shi, Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, inaccordance with the Budapest Treaty for the Deposit of Microorganisms,and was accorded the accession number FERM BP-7555, FERM BP-7557, FERMBP-7558, and FERM BP-7559, respectively

[0597] (3) Construction of Expression Plasmids Carrying the Heavy ChainVariable Region DNA of Several Types Humanized or Mouse TRA-8

[0598] Recombinant expression vector for animal cells are constructed byinserting the DNA encoding the heavy chain of H1 type, H3 type, and H4type humanized or M type chimeric TRA-8 (cloned in above) as follows.

[0599] One μg of plasmid pSRHHH3 (European patent application EP 0 909816 A1) carrying the heavy chain variable region of humanized anti-Fasmonoclonal antibody HFE7A and human IgG1 constant region genomic DNA, anexpression vector for mammalian cells, is digested with the restrictionenzymes HindIII and ApaI, and separated by 3% agarose gelelectrophoresis. After electrophoresis, the gel is stained with a 1μg/ml aqueous solution of ethidium bromide to allow detection of DNAunder UV light. The vector DNA bands containing human IgG1 constantregion genomic DNA without the heavy chain variable region of humanizedHFE7A are cut out using a razor blade and eluted from the gel usingGeneclean Spin Kit. After phenol extraction, the eluted DNA is thenconcentrated by centrifugation at 7,500×g, followed by ethanolprecipitation, and finally dissolved in 5 μl of distilled water and thendephosphorylated using CIP. The resulting digested, dephosphorylatedplasmid (100 ng) is ligated with 1 μg of the DNA fragment of pHA15,pHC10, pHD21, or pM11 containing the DNA encoding the heavy chainvariable region of humanized or chimeric TRA-8, which bad also beendigested with HindIII and ApaI, using a DNA Ligation Kit Version 2.0(Takara Shuzo Co., Ltd.). The ligation mixture is then used to transformE. coli JM109, which is then plated on LB agar plates containing 50μg/ml ampicillin.

[0600] The transformants obtained by this method are cultured in 2 ml ofliquid LB medium containing 50 μg/ml ampicillin at 37° C. overnight, andplasmid DNA is subsequently extracted from the resulting culture by thealkaline-SDS method.

[0601] The extracted plasmid DNA is digested with HindIII and ApaI, andsubjected to 3% w/v agarose gel electrophoresis to confirm the presenceor absence of the insert of the DNA encoding the heavy chain variableregion of humanized or chimeric TRA-8. The insertion and orientation ofthe desired DNA fragment in the vector is confirmed by DNA sequencingusing a gene sequence analyzer (ABI Prism 3700 DNA Analyzer; AppliedBiosystems). The resulting expression plasmids carrying cDNA encodingthe heavy chain of humanized or chimeric TRA-8 were designated pHA15-1,pHC10-3, pHD21-1, and pM11-1, respectively.

[0602] (4) Construction of Vectors for the Humanized Light Chains

[0603] (4.1) Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody (LM1 Type)

[0604] As shown in SEQ ID No. 72 of the Sequence Listing, otherhumanization (LM1 type) of the amino acid sequences of the light chainof the mouse anti-human DR5 antibody TRA-8 entailed replacing the 3rdamino acid (valine), 8th amino acid (histidine), 9th amino acid(lysine), 10th amino acid (phenylalanine), 11th amino acid (methionine),13th amino acid (threonine), 20th amino acid (serine), 42nd amino acid(glutamine), 43rd (serine), 60th amino acid (aspartic acid), 63rd aminoacid (threonine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 85th aminoacid (aspartic acid), 87th amino acid (phenylalanine),and 99th aminoacid (glycine) 103rd amino acid (leucine) and 108th amino acid (alanine)from the N-terminus of the amino acid sequence of the TRA-8 light chainare replaced with glutamine, proline, serine, serine, leucine, alanine,threonine, lysine, alanine, serine, serine, serine, leucine, proline,phenylalanine, threonine, tyrosine, glutamine, valine and threoninerespectively. The resulting sequence is designated LM1.

[0605] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 (LM1 type, SEQID No. 72 of the Sequence Listing) are constructed as follows.

[0606] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized TRA-8 (LM1 Type)

[0607] DNA coding for the LM1 polypeptide chain (SEQ ID No. 72 of theSequence Listing), each of which is a fusion of the variable region ofhumanized anti-DR5 antibody TRA-8 light chain (LM1 type) and theconstant region of the human Ig light chain (K chain), are respectivelysynthesized by using combinations of PCR.

[0608] Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), HKCDF11(SEQ ID No. 50), HKCDR12 (SEQ ID No. 51), HKCDF22 (SEQ ID No. 52),HKCDR22 (SEQ ID No. 53), and HKCF12 (SEQ ID No. 54).

[0609] The following oligonucleotide primers are synthesized for PCR:5′-gtcccccaca gatgcagaca aagaacttgg agattgggtc atctgaatgt caccagtgga-3′.(HKSPR12; SEQ ID No. 77)

[0610] 2) Construction of Plasmid pCR3.1/LM1-2 (Cloning of HumanizedTRA-8 Light Chain Type LM1)

[0611] LM1-DNA fragment coding for the amino acid sequence as defined inSEQ ID No. 72 of the same is prepared by performing 2-step PCR, insertedinto a plasmid vector and cloned in E. coli.

[0612] a) First Step PCR

[0613] LM1-F1-DNA fragment coding for a secretion signal sequence and aportion of FRL₁ region with a Hind III restriction enzyme cleavage siteadded at the 5′-end is prepared under the following conditions. Thetemplate plasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0909816 A1.

[0614] Composition of the reaction solution:

[0615] plasmid pHSGHM17 DNA, 25 ng

[0616] oligonucleotide primer 7AL1P, 50 pmol

[0617] oligonucleotide primer HKSPR12, 50 pmol

[0618] dNTPs cocktail, 5 μl

[0619] 10×PCR buffer, 5 μl

[0620] ampliTaq DNA polymerase (PerkinElmer), 2.5 units

[0621] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0622] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0623] LM1-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂,and CDRL₂ is prepared under the following conditions.

[0624] Composition of the reaction solution:

[0625] plasmid pL28 DNA, 25 ng

[0626] oligonucleotide primer HKCDF11, 50 pmol

[0627] oligonucleotide primer HKCDR12, 50 pmol

[0628] dNTPs cocktail, 5 μl

[0629] 10×PCR buffer, 5 μl

[0630] ampliTaq DNA polymerase, 2.5 units

[0631] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0632] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0633] LM1-F3-DNA fragment coding for CDRL2, FRL₃, and a portion ofCDRL₃ is prepared under the following conditions.

[0634] Composition of the reaction solution:

[0635] plasmid pSRPDHH DNA, 25 ng

[0636] oligonucleotide primer HKCDF22, 50 pmol

[0637] oligonucleotide primer HKCDR22, 50 pmol

[0638] dNTPs cocktail, 5 μl

[0639] 10×PCR buffer, 5 μl

[0640] ampliTaq DNA polymerase, 2.5 units

[0641] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0642] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0643] LM1-F4-DNA fragment coding for CDRL₃, FRL₄ and the constantregion with an EcoR I restriction enzyme cleavage site added at the3′-end is prepared under the following conditions.

[0644] Composition of the reaction solution:

[0645] plasmid pSRPDHH DNA, 25 ng

[0646] oligonucleotide primer HKCF12, 50 pmol

[0647] oligonucleotide primer 7ALCN, 50 pmol

[0648] dNTPs cocktail, 5 μl

[0649] 10×PCR buffer, 5 μl

[0650] ampliTaq DNA polymerase, 2.5 units

[0651] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0652] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0653] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor

[0654] b) Second Step PCR

[0655] LM1-DNA in which above described LM1-F1-DNA, LM1-F2-DNA,LM1-F3-DNA and LM1-F4-DNA fragments are fused is prepared under thefollowing conditions.

[0656] Composition of the reaction solution:

[0657] Gel fragment of LM1-F1-DNA prepared in the first step PCR,

[0658] Gel fragment of LM1-F2-DNA prepared in the first step PCR,

[0659] Gel fragment of LM1-F3-DNA prepared in the first step PCR,

[0660] Gel fragment of LM 1-F4-DNA prepared in the first step PCR

[0661] oligonucleotide primer 7AL1P, 50 pmol

[0662] oligonucleotide primer 7ALCN, 50 pmol

[0663] dNTPs cocktail, 5.0 μl

[0664] 10×PCR buffer, 5.0 ¹l

[0665] ampliTaq DNA polymerase, 2.5 units

[0666] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0667] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0668] The thus prepared LM 1-DNA fragment is inserted into plasmidpCR3.1 DNA using Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 (LM1 type) are confirmed bythe dideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad.Sci. USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin ElmerApplied Biosystems, Japan).

[0669] The resulting plasmids are designated pCR3.1/LM1-2 (the plasmidcarrying cDNA encoding the light chain variable region of humanizedTRA-8 (LM1 type) and a human Ig light chain constant region).

[0670] The obtained plasmid pCR3.1/LM1-2 containing LM1-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

[0671] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM1-DNA fragment,that had been digested with the restriction enzymes Hind III and EcoR I,are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM1-DNA fragment is selected by 1%agarose gel electrophoresis.

[0672] As a result of the above procedure, plasmid pHSG/M1-2-2 carryinga fusion fragment of the variable region of the humanized LM1 TRA-8light chain and the constant region of human IgK chain is obtained. Thetransformant E coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M1-2-2 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7562.

[0673] 3) Construction of Plasmid pSR/LM1-2 (Expression Plasmid forHumanized LM1 TRA-8 Light Chain)

[0674] The obtained plasmid pHSG/M1-2-carrying a fusion fragment of thevariable region of the humanized LM1 TRA-8 light chain and the constantregion of human IgK chain is digested with the restriction enzymes HindIII and EcoR I.

[0675] One μg of cloning plasmid pSRPDHH DNA (European patentapplication EP 0 909 816 A1) is digested with the restriction enzymesHind III and EcoR I, and then dephosphorylated with CIP. The resultingdephosphorylated pSRPDHH DNA and HindIII-EcoRI DNA fragment obtainedfrom pHSG/M1-2-2 are ligated using DNA Ligation Kit Version 2.0 (TakaraSyuzo, Co. Ltd.). Then, E. coli DH5α is transformed with the ligated DNAand spread onto LB agar. The transformants obtained are cultured inliquid LB medium containing 100 μg/ml ampicillin, and plasmid DNA isextracted from the resulting culture according to the alkaline-SDSmethod. The insertion and orientation of the desired DNA fragment inpSRPDHH vector is confirmed by DNA sequencing using a gene sequenceanalyzer.

[0676] The resulting expression plasmid carrying cDNA encoding the lightchain of humanized LM1 TRA-8 is designated pSR/LM1-2.

[0677] (4.2) Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody (LM3 type)

[0678] As shown in SEQ ID No. 73 of the Sequence Listing, otherhumanization (LM3 type) of the amino acid sequences of the light chainof the mouse anti-human DR5 antibody TRA-8 entailed replacing the 8thamino acid (histidine), 9th amino acid (lysine), 10th amino acid(phenylalanine), 11th amino acid (methionine), 13th amino acid(threonine), 20th amino acid (serine), 42nd amino acid (glutamine), 43rdamino acid (serine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 85th aminoacid (aspartic acid), 87th amino acid (phenylalanine), 99th amino acid(glycine) 103rd amino acid (leucine) and 108th amino acid (alanine) fromthe N-terminus of the amino acid sequence of the TRA-8 light chain arereplaced with proline, serine, serine, leucine, alanine, threonine,lysine, alanine, serine, leucine, proline, phenylalanine, threonine,tyrosine, glutamine, valine and threonine, respectively. The resultingsequence is designated LM3.

[0679] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 (LM3 type, SEQID No. 73 of the Sequence Listing) are constructed as follows.

[0680] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized LM3 TRA-8

[0681] DNA coding for the LM3 polypeptide chain (SEQ ID No. 73 of theSequence Listing), each of which is a fusion of the variable region ofhumanized anti-DR5 antibody TRA-8 light chain and the constant region ofthe human Ig light chain (K chain), are respectively synthesized byusing combinations of PCR.

[0682] Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR: 5′-atctagttctcagagatgga gacagacaca atcctgctat gggtgctgct gctctgggtt ccagg-3′;(MOD1F1; SEQ ID No. 78) 5′-cagcacccat agcaggattg tgtctgtctc catctctgagaactagatga gaggatgctt (MOD1R1; SEQ ID No. 79) cttaagctt-3′;5′-ctccactggt gacattgtga tgacccaatc tccaagttct ttgtctgcat ctgtggggga(MOD1F22; SEQ ID No. 80) cagggtc-3′; 5′-acttggagat tgggtcatca caatgtcaccagtggagcct ggaacccaga gcag-3′ (MOD1R22; SEQ ID No. 81) 5′-accatcacctgcaaggccag tcaggatgtg ggtactgctg tagcctggta ccaacagaaa (MOD1F3; SEQ IDNo. 82) ccaggaa-3′; 5′-tacagcagta cccacatcct gactggcctt gcaggtgatggtgaccctgt cccccacaga (MOD1R3; SEQ ID No. 83) tgcagacaaa ga-3′;5′-aagcacccaa actcctcatc tattgggcat ccacccggca cactggggtc ccagataggt(MOD1F42; SEQ ID No. 84) ttacaggcag t-3′; 5′-cccagtgtgc cgggtggatgcccaatagat gaggagtttg ggtgcttttc ctggtttctg (MOD1R4; SEQ ID No. 85)ttggtaccag gc-3′; 5′-gggtctggga cagacttcac cctcaccatc tctagtctgcagccggagga ttttgcaacc tat-3′; (MOD1F5; SEQ ID No. 86) 5′-actagagatggtgagggtga agtctgtccc agacccactg cctgtaaacc tatctgggac-3′; (MOD1R52; SEQID No. 87) 5′-tactgtcagc aatatagcag ctatcggacg ttcggtcaag gcaccaaggtggaaatc-3′; (MOD1F6; SEQ ID No. 88) 5′-cgtccgatag ctgctatatt gctgacagtaataggttgca aaatcctccg gctgcac-3′ (MOD1R6; SEQ ID No. 89) 5′-aaacggactgtggctgcacc atctgtcttc atcttcccgc catctgatga g-3′; (MOD1F7; SEQ ID No.90) 5′-gaagatgaag acagatggtg cagccacagt ccgtttgatt tccaccttgg tgccttgaccgaa-3′; (MOD1R7; SEQ ID No. 91) and 5′-agatttcaac tgctcatcag atggcgggaa.(LR17; SEQ ID No. 101)

[0683] 2) Construction of Plasmid pCR3.1/LM3-3-44 (Cloning of HumanizedTRA-8 Light Chain Type LM3)

[0684] LM3-DNA fragment coding for the amino acid sequence as defined inSEQ ID No. 73 of the same is prepared by performing 2-step PCR, insertedinto a plasmid vector and cloned in E. coli.

[0685] a) First Step PCR

[0686] LM3-F31B-DNA fragment coding for a secretion signal sequenceregion with a Hind III restriction enzyme cleavage site added at the5′-end, FRL₁ CDRL₁, FRL₂, and CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion ofthe constant region is prepared under the following conditions.

[0687] Composition of the reaction solution:

[0688] oligonucleotide primer MOD1F1, 5 pmol

[0689] oligonucleotide primer MOD1R1, 5 pmol

[0690] oligonucleotide primer MOD1F22, 5 pmol

[0691] oligonucleotide primer MOD1R22, 5 pmol

[0692] oligonucleotide primer MOD1F3, 5 pmol

[0693] oligonucleotide primer MOD1R3, 5 pmol

[0694] oligonucleotide primer MOD1F42, 5 pmol

[0695] oligonucleotide primer MOD1R4, 5 pmol

[0696] oligonucleotide primer MOD1F5, 5 pmol

[0697] oligonucleotide primer MOD1R52, 5 pmol

[0698] oligonucleotide primer MOD1F6, 5 pmol

[0699] oligonucleotide primer MOD1R6, 5 pmol

[0700] oligonucleotide primer MOD1F7, 50 pmol

[0701] oligonucleotide primer MOD1R7, 5 pmol

[0702] oligonucleotide primer 7AL1P, 50 pmol

[0703] oligonucleotide primer LR17, 50 pmol

[0704] dNTPs cocktail, 5 μl

[0705] 10×PCR buffer, 5 μl

[0706] ampliTaq DNA polymerase, 2.5 units

[0707] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0708] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0709] LM3-F31C-DNA fragment coding for a portion of the constant regionwith an Eco R I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

[0710] The template plasmids, pSRPDHH, is obtained by following thedescription in a European patent application EP 0 909 816 A1.

[0711] Composition of the reaction solution:

[0712] plasmid pSRPDHH DNA, 25 ng

[0713] oligonucleotide primer MOD1F7, 50 pmol

[0714] oligonucleotide primer 7ALCN, 50 pmol

[0715] dNTPs cocktail, 5 μl

[0716] 10×PCR buffer, 5 μl

[0717] ampliTaq DNA polymerase, 2.5 units

[0718] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0719] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0720] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor.

[0721] b) Second Step PCR

[0722] LM3-DNA in which above described LM3-F31B-DNA, and LM3-F31C-DNAfragments are fused is prepared under the following conditions.Composition of the reaction solution:

[0723] Gel fragment of LM3-F31B-DNA prepared in the first step PCR,

[0724] Gel fragment of LM3-F31C-DNA prepared in the first step PCR,

[0725] oligonucleotide primer 7AL1P, 50 pmol

[0726] oligonucleotide primer 7ALCN, 50 pmol

[0727] dNTPs cocktail, 5.0 μl

[0728] 10×PCR buffer, 5.0 μl

[0729] ampliTaq DNA polymerase, 2.5 units

[0730] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0731] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0732] The thus prepared LM3-DNA fragment is inserted into plasmidpCR3.1DNA using Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM3 TRA-8 are confirmed by thedideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin ElmerApplied Biosystems, Japan).

[0733] The resulting plasmids are designated pCR3.1/LM3-3-44 (theplasmid carrying cDNA encoding the light chain variable region ofhumanized LM3 TRA-8 and a human Ig light chain constant region).

[0734] The obtained plasmid pCR3.1/LM3-3-44 containing LM3-DNA fragmentis digested with the restriction enzymes Hind III and EcoR I.

[0735] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM3-DNA fragment,that had been digested with the restriction enzymes Hind III and EcoR I,are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α. is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM3-DNA fragment is selected by 1%agarose gel electrophoresis.

[0736] As a result of the above procedure, plasmid pHSG/M3-3-22 carryinga fusion fragment of the variable region of the humanized LM3 TRA-8light chain and the constant region of human IgK chain is obtained. Thetransformant E coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M3-3-22 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7564.

[0737] 3) Construction of Plasmid pSR/LM3-3-44-10 (Expression Plasmidfor Humanized LM3 TRA-8 Light Chain)

[0738] The obtained plasmid pHSG/M3-3-22 carrying a fusion fragment ofthe variable region of the humanized LM3 TRA-8 light chain and theconstant region of human IgK chain is digested with the restrictionenzymes Hind III and EcoR I.

[0739] One μg of cloning plasmid pSRPDHH DNA (European patentapplication EP 0 909 816 A1) is digested with the restriction enzymesHind III and EcoR I, and then dephosphorylated with CIP. The resultingdephosphorylated pSRPDHH DNA and HindIII-EcoRI DNA fragment obtainedfrom pHSG/M3-3-22 are ligated using DNA Ligation Kit Version 2.0 (TakaraSyuzo, Co. Ltd.). Then, E. Coli DH5α is transformed with the ligated DNAand spread onto LB agar. The transformants obtained are cultured inliquid LB medium containing 100 μg/ml ampicillin, and plasmid DNA isextracted from the resulting culture according to the alkaline-SDSmethod. The insertion and orientation of the desired DNA fragment inpSRPDHH vector is confirmed by DNA sequencing using a gene sequenceanalyzer (ABI Prism 3700 DNA Analyzer; Applied Biosystems).

[0740] The resulting expression plasmid carrying cDNA encoding the lightchain of humanized LM3 TRA-8 is designated pSR/LM3-3-44-10.

[0741] (4.3) Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody (LM4 Type)

[0742] As shown in SEQ ID No. 74 of the Sequence Listing, otherhumanization (LM4 type) of the amino acid sequences of the light chainof the mouse anti-human DR5 antibody TRA-8 entailed replacing the 8thamino acid (histidine), 9th amino acid (lysine), 10th amino acid(phenylalanine), 11th amino acid (methionine), 13th amino acid(threonine), 20th amino acid (serine), 42nd amino acid (glutamine), 43rdamino acid (serine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 85th aminoacid (aspartic acid), 99th amino acid (glycine) 103rd amino acid(leucine) and 108th amino acid (alanine) from the N-terminus of theamino acid sequence of the TRA-8 light chain are replaced with proline,serine, serine, leucine, alanine, threonine, lysine, alanine, serine,leucine, proline, phenylalanine, threonine, glutamine, valine andthreonine respectively. The resulting sequence is designated LM4.

[0743] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 (LM4 type)(SEQ ID No. 74 of the Sequence Listing) are constructed as follows.

[0744] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized LM4 TRA-8

[0745] DNA coding for the LM4 polypeptide chain (SEQ ID No. 74 of theSequence Listing), each of which is a fusion of the variable region ofhumanized anti-DR5 antibody TRA-8 light chain and the constant region ofthe human Ig light chain (K chain), are respectively synthesized byusing combinations of PCR.

[0746] Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), MOD1F1(SEQ ID No. 78), MOD1R1 (SEQ ID No. 79), MOD1F22 (SEQ ID No. 80),MOD1R22 (SEQ ID No. 81), MOD1F3 (SEQ ID No. 82), MOD1R3 (SEQ ID No. 83),MOD1F42 (SEQ ID No. 84), MOD1R4 (SEQ ID No. 85), MOD1F5 (SEQ ID No. 86),MOD1R52 (SEQ ID No. 87), MOD1F7 (SEQ ID No. 90), and MOD1R7 (SEQ ID No.91), LR17 (SEQ ID No. 101), the following oligonucleotide primers aresynthesized for PCR: 5′-ttctgtcagc aatatagcag ctatcggacg ttcggtcaaggcaccaaggt ggaaatc-3′ (MOD1F62; SEQ ID No. 92) 5′-cgtccgatag ctgctatattgctgacagaa ataggttgca aaatcctccg gctgcag-3′ (MOD1R62; SEQ ID No. 93)

[0747] 2) Construction of Plasmid pCR3.1/LM4-5-3 (Cloning of HumanizedTRA-8 Light Chain Type LM4)

[0748] LM4-DNA fragment coding for the amino acid sequence as defined inSEQ ID No. 74 of the same is prepared by performing 2-step PCR, insertedinto a plasmid vector and cloned in E. coli.

[0749] a) First Step PCR

[0750] LM4-F41B-DNA fragment coding for a secretion signal sequenceregion with a Hind III restriction enzyme cleavage site added at the5′-end, FRL₁, CDRL₁, FRL₂, and CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion ofthe constant region is prepared under the following conditions.

[0751] Composition of the reaction solution:

[0752] oligonucleotide primer MOD1F1, 5 pmol

[0753] oligonucleotide primer MOD1R1, 5 pmol

[0754] oligonucleotide primer MOD1F22, 5 pmol

[0755] oligonucleotide primer MOD1R22, 5 pmol

[0756] oligonucleotide primer MOD1F3, 5 pmol

[0757] oligonucleotide primer MOD1R3, 5 pmol

[0758] oligonucleotide primer MOD1F42, 5 pmol

[0759] oligonucleotide primer MOD1R4, 5 pmol

[0760] oligonucleotide primer MOD1F5, 5 pmol

[0761] oligonucleotide primer MOD1R52, 5 pmol

[0762] oligonucleotide primer MOD1F62, 5 pmol

[0763] oligonucleotide primer MOD1R62, 5 pmol

[0764] oligonucleotide primer MOD1F7, 50 pmol

[0765] oligonucleotide primer MOD1R7, 5 pmol

[0766] oligonucleotide primer 7AL1P, 50 pmol

[0767] oligonucleotide primer LR17, 50 pmol

[0768] dNTPs cocktail, 5 μl

[0769] 10×PCR buffer, 5 μl

[0770] ampliTaq DNA polymerase, 2.5 units

[0771] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0772] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0773] LM4-F41 C-DNA fragment coding for a portion of the constantregion with an Eco R I restriction enzyme cleavage site added at the3′-end is prepared under the following conditions.

[0774] The template plasmids, pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

[0775] Composition of the reaction solution:

[0776] plasmid pSRPDHH DNA, 25 ng

[0777] oligonucleotide primer MOD 1F7, 50 pmol

[0778] oligonucleotide primer 7ALCN, 50 pmol

[0779] dNTPs cocktail, 5 μl

[0780] 10×PCR buffer, 5 μl

[0781] ampliTaq DNA polymerase, 2.5 units

[0782] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0783] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0784] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor blade.

[0785] b) Second Step PCR

[0786] LM4-DNA in which above described LM4-F41B-DNA, and LM4-F41C-DNAfragments are fused is prepared under the following conditions.

[0787] Composition of the reaction solution:

[0788] Gel fragment of LM4-F41B-DNA prepared in the first step PCR,

[0789] Gel fragment of LM4-F41 C-DNA prepared in the first step PCR,

[0790] oligonucleotide primer 7AL1P, 50 pmol

[0791] oligonucleotide primer 7ALCN, 50 pmol

[0792] dNTPs cocktail, 5.0 μl

[0793] 10×PCR buffer, 5.0 μl

[0794] ampliTaq DNA polymerase, 2.5 units

[0795] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0796] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0797] The thus prepared LM4-DNA fragment is inserted into plasmidpCR3.1DNA using Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM4 TRA-8 are confirmed by thedideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM; Perkin ElmerApplied Biosystems, Japan).

[0798] The resulting plasmids are designated pCR3.1/LM4-5-3 (the plasmidcarrying cDNA encoding the light chain variable region of humanized LM4TRA-8 and a human Ig light chain constant region).

[0799] The obtained plasmid pCR3.1/LM4-5-3 containing LM4-DNA fragmentis digested with the restriction enzymes Hind III and EcoR I.

[0800] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM4-DNA fragment,that had been digested with the restriction enzymes Hind III and EcoR I,are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM4-DNA fragment is selected by 1%agarose gel electrophoresis.

[0801] As a result of the above procedure, plasmid pHSG/M4-5-3-1carrying a fusion fragment of the variable region of the humanized LM4TRA-8 light chain and the constant region of human IgK chain isobtained. The transformant E coli strain harboring these plasmid,designated as E. coli DH5α/pHSG/M4-5-3-1 was deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, 1-1, Higashi 1 chome Tsukuba-shi,Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, in accordance with theBudapest Treaty for the Deposit of Microorganisms, and was accorded theaccession number FERM BP-7565.

[0802] 3) Construction of Plasmid pSR/LM4-5-3-3 (Expression Plasmid forHumanized LM4 TRA-8 Light Chain)

[0803] The obtained plasmid pHSG/M4-5-3-1 carrying a fusion fragment ofthe variable region of the humanized LM4 TRA-8 light chain and theconstant region of human IgK chain is digested with the restrictionenzymes Hind III and EcoR I.

[0804] One μg of cloning plasmid pSRPDHH DNA (European patentapplication EP 0 909 816 A1) is digested with the restriction enzymesHind III and EcoR I, and then dephosphorylated with CIP. The resultingdephosphorylated pSRPDHH DNA and HindIII-EcoRi DNA fragment obtainedfrom pHSG/M4-5-3-1 are ligated using DNA Ligation Kit Version 2.0(Takara Syuzo, Co. Ltd.). Then, E. coli DH5α is transformed with theligated DNA and spread onto LB agar. The transformants obtained arecultured in liquid LB medium containing 100 μg/ml ampicillin, andplasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The insertion and orientation of the desired DNAfragment in pSRPDHH vector is confirmed by DNA sequencing using a genesequence analyzer (ABI Prism 3700 DNA Analyzer; Applied Biosystems).

[0805] The resulting expression plasmid carrying cDNA encoding the lightchain of humanized LM4 TRA-8 is designated pSR/LM4-5-3-3.

[0806] (4.4) Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody (LM5 Type)

[0807] As shown in SEQ ID No. 75 of the Sequence Listing, otherhumanization (LM5 type) of the amino acid sequences of the light chainof the mouse anti-human DR5 antibody TRA-8 entailed replacing the 8thamino acid (histidine), 9th amino acid (lysine), 10th amino acid(phenylalanine), 11th amino acid (methionine), 13th amino acid(threonine), 20th amino acid (serine), 42nd amino acid (glutamine), 43rdamino acid (serine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 103rdamino acid (leucine) and 108th amino acid (alanine) from the N-terminusof the amino acid sequence of the TRA-8 light chain are replaced withproline, serine, serine, leucine, alanine, threonine, lysine, alanine,serine, leucine, proline, phenylalanine, valine and threoninerespectively. The resulting sequence is designated LM5.

[0808] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 (LM5 type)(SEQ ID No. 75 of the Sequence Listing) is constructed as follows.

[0809] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized LM5 TRA-8

[0810] DNA coding for the LM5 polypeptide chain (SEQ ID No. 75 of theSequence Listing), each of which is a fusion of the variable region ofhumanized anti-DR5 antibody TRA-8 light chain and the constant region ofthe human Ig light chain (K chain), are respectively synthesized byusing combinations of PCR.

[0811] Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), MOD1F1(SEQ ID No. 78), MOD1R1 (SEQ ID No. 79), MOD1F22 (SEQ ID No. 80),MOD1R22 (SEQ ID No. 81), MOD1F3 (SEQ ID No. 82), MOD1R3 (SEQ ID No. 83),MOD1F42 (SEQ ID No. 84), MOD1R4 (SEQ ID No. 85), MOD1R52 (SEQ ID No.87), MOD1F7 (SEQ ID No. 90), and LR17 (SEQ ID No. 101), the followingoligonucleotide primers are synthesized for PCR: 5′-gggtctgggacagacttcac cctcaccatc tctagtctgc agccggagga ttttgcagat tat-3′ (MOD1F52;SEQ ID No. 94) 5′-ttctgtcagc aatatagcag ctatcggacg ttcggtggag gcaccaaggtggaaatc-3′ (MOD1F63; SEQ ID No. 95) 5′-cgtccgatag ctgctatatt gctgacagaaataatctgca aaatcctccg gctgcag-3′ (MOD1R63; SEQ ID No. 96) 5′-gaagatgaagacagatggtg cagccacagt ccgtttgatt tccaccttgg tgcctccacc gaa-3′ (MOD1R72;SEQ ID No. 102)

[0812] 2) Construction of Plasmid pCR3.1/LM5-3-42 (Cloning of HumanizedTRA-8 Light Chain Type LM5)

[0813] LM5-DNA fragment coding for the amino acid sequence as defined inSEQ ID No. 75 of the same is prepared by performing 2-step PCR, insertedinto a plasmid vector and cloned in E. coli.

[0814] a) First Step PCR

[0815] LM5-F51B-DNA fragment coding for a secretion signal sequenceregion with a Hind III restriction enzyme cleavage site added at the5′-end, FRL₁, CDRL₁, FRL₂, CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion of theconstant region is prepared under the following conditions.

[0816] Composition of the reaction solution:

[0817] oligonucleotide primer MOD1F1, 5 pmol

[0818] oligonucleotide primer MOD1R1, 5 pmol

[0819] oligonucleotide primer MOD1F22, 5 pmol

[0820] oligonucleotide primer MOD1R22, 5 pmol

[0821] oligonucleotide primer MOD1F3, 5 pmol

[0822] oligonucleotide primer MOD1R3, 5 pmol

[0823] oligonucleotide primer MOD1F42, 5 pmol

[0824] oligonucleotide primer MOD1R4, 5 pmol

[0825] oligonucleotide primer MOD1F52, 5 pmol

[0826] oligonucleotide primer MOD1R52, 5 pmol

[0827] oligonucleotide primer MOD1F63, 5 pmol

[0828] oligonucleotide primer MOD1R63, 5 pmol

[0829] oligonucleotide primer MOD1F7, 50 pmol

[0830] oligonucleotide primer MOD1R72, 5 pmol

[0831] oligonucleotide primer 7AL1P, 50 pmol

[0832] oligonucleotide primer LR17, 50 pmol

[0833] dNTPs cocktail, 5 μl

[0834] 10×PCR buffer, 5 μl

[0835] ampliTaq DNA polymerase, 2.5 units

[0836] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0837] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0838] LM5-F51C-DNA fragment coding for a portion of the constant regionwith an Eco R I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions. The template plasmids, pSRPDHH,is obtained by following the description in an European patentapplication EP 0 909 816 A1.

[0839] Composition of the reaction solution:

[0840] plasmid pSRPDHH DNA, 25 ng

[0841] oligonucleotide primer MOD1F7, 50 pmol

[0842] oligonucleotide primer 7ALCN, 50 pmol

[0843] dNTPs cocktail, 5 μl

[0844] 1 ×PCR buffer, 5 μl

[0845] ampliTaq DNA polymerase, 2.5 units

[0846] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0847] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0848] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor blade.

[0849] b) Second Step PCR

[0850] LM5-DNA in which above described LM5-F51B-DNA, and LM5-F51C-DNAfragments are fused is prepared under the following conditions.Composition of the reaction solution:

[0851] Gel fragment of LM5-F51B-DNA prepared in the first step PCR,

[0852] Gel fragment of LM5-F51 C-DNA prepared in the first step PCR,

[0853] oligonucleotide primer 7AL1P, 50 pmol

[0854] oligonucleotide primer 7ALCN, 50 pmol

[0855] dNTPs cocktail, 5.0 μl

[0856] 1×PCR buffer, 5.0 μl

[0857] ampliTaq DNA polymerase, 2.5 units

[0858] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0859] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0860] The thus prepared LM5-DNA fragment is inserted into plasmidpCR3.1DNA using Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM5 TRA-8 are confirmed by thedideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using DNA analyzer.

[0861] The resulting plasmids are designated pCR3.1/LM5-3-42 (theplasmid carrying cDNA encoding the light chain variable region ofhumanized LM5 TRA-8 and a human Ig light chain constant region).

[0862] The obtained plasmid pCR3.1/LM5-3-42 containing LM5-DNA fragmentis digested with the restriction enzymes Hind III and EcoR I.

[0863] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM5-DNA fragment,that had been digested with the restriction enzymes Hind III and EcoR I,are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM5-DNA fragment is selected by 1%agarose gel electrophoresis.

[0864] As a result of the above procedure, plasmid pHSG/M5-3-27 carryinga fusion fragment of the variable region of the humanized LM5 TRA-8light chain and the constant region of human IgK chain is obtained.

[0865] 3) Construction of Plasmid pSR/LM5-3-27-1 (Expression Plasmid forHumanized LM5 TRA-8 Light Chain)

[0866] The obtained plasmid pHSG/M5-3-27 carrying a fusion fragment ofthe variable region of the humanized LM5 TRA-8 light chain and theconstant region of human Igκ chain is digested with the restrictionenzymes Hind III and EcoR I.

[0867] One μg of cloning plasmid pSRPDHH DNA (European patentapplication EP 0 909 816 A1) is digested with the restriction enzymesHind III and EcoR I, and then dephosphorylated with CIP. The resultingdephosphorylated pSRPDHH DNA and HindIII-EcoRI DNA fragment obtainedfrom pHSG/M5-3-27 are ligated using DNA Ligation Kit Version 2.0 (TakaraSyuzo, Co. Ltd.). Then, E. coli DH5α is transformed with the ligated DNAand spread onto LB agar. The transformants obtained are cultured inliquid LB medium containing 100 μg/ml ampicillin, and plasmid DNA isextracted from the resulting culture according to the alkaline-SDSmethod. The insertion and orientation of the desired DNA fragment inpSRPDHH vector is confirmed by DNA sequencing using a gene sequenceanalyzer (ABI Prism 3700 DNA Analyzer; Applied Biosystems).

[0868] The resulting expression plasmid carrying cDNA encoding the lightchain of humanized LM5 TRA-8 is designated pSR/LM5-3-27-1.

[0869] (4.5) Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody (Chimera Type)

[0870] The sequence shown in SEQ ID No. 76 of the Sequence Listing, theamino acid sequence of the light chain of chimera type TRA-8, isdesignated LM6.

[0871] Expression plasmids carrying this type of humanized light chainamino acid sequences of the anti-human DR5 antibody TRA-8 (LM6 type)(SEQ ID No. 75 of the Sequence Listing) is constructed as follows.

[0872] 1) Synthesis of Primers for Preparing the Variable and ConstantRegions of the Light Chain of Humanized LM6 TRA-8

[0873] DNA coding for the LM6 polypeptide chain (SEQ ID No. 75 of theSequence Listing), each of which is a fusion of the variable region ofmouse anti-DR5 antibody TRA-8 light chain (LM6 type) and the constantregion of the human Ig light chain (K chain), are respectivelysynthesized by using combinations of PCR.

[0874] Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR: 5′-tgatgtggacatgaatttgt gagactgggt catcacaatg tcaccagtgg a-3′; (HKSPR13; SEQ ID No.97) 5′-tgggttccag gctccactgg tgacattgtg atgacccagt ctcacaaatt c-3′;(MVF11; SEQ ID No. 98) 5′-aagacagatg gtgcagccac agcccgtttg atttccagcttggtgcctc-3′; and (MVR11; SEQ ID No. 99) 5′-aagctggaaa tcaaacgggctgtggctgca ccatctgtct tcatc-3′. (MCF11; SEQ ID No. 100)

[0875] 2) Construction of Plasmid pCR3.1/LM6-1-16 (Cloning of HumanizedTRA-8 Light Chain Type LM6)

[0876] LM6-DNA fragment coding for the amino acid sequence, as definedin SEQ ID No. 75 of the same is prepared by performing 2-step PCR,inserted into a plasmid vector and cloned in E. coli.

[0877] a) First Step PCR

[0878] LM6-FI-DNA fragment coding for a secretion signal sequence and aportion of FRL₁ region with a Hind III restriction enzyme cleavage siteadded at the 5′-end is prepared under the following conditions. Thetemplate plasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

[0879] Composition of the reaction solution:

[0880] plasmid pHSGHM17 DNA, 25 ng

[0881] oligonucleotide primer 7AL1P, 50 pmol

[0882] oligonucleotide primer HKSPR13, 50 pmol

[0883] dNTPs cocktail, 5 μl

[0884] 10×PCR buffer, 5 μl

[0885] ampliTaq DNA polymerase (PerkinElmer), 2.5 units

[0886] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0887] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0888] LM6-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂,CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion of the constant region isprepared under the following conditions.

[0889] Composition of the reaction solution:

[0890] plasmid pL28 DNA, 25 ng

[0891] oligonucleotide primer MVF11, 50 pmol

[0892] oligonucleotide primer MVR12, 50 pmol

[0893] dNTPs cocktail, 5 μl

[0894] 10×PCR buffer, 5 μl

[0895] ampliTaq DNA polymerase, 2.5 units

[0896] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0897] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0898] LM6-F3-DNA fragment coding for a portion of FRL₄ and the constantregion with an EcoR I restriction enzyme cleavage site added at the3′-end is prepared under the following conditions.

[0899] Composition of the reaction solution:

[0900] plasmid pSRPDHH DNA, 25 ng

[0901] oligonucleotide primer MCF11, 50 pmol

[0902] oligonucleotide primer 7ALCN, 50 pmol

[0903] dNTPs cocktail, 5 μl

[0904] 10×PCR buffer, 5 μl

[0905] ampliTaq DNA polymerase, 2.5 units

[0906] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0907] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0908] The amplified DNA fragments after PCR are separated by 5%polyacrylamide gel electrophoresis. The gel after electrophoresis isstained with 1 μg/ml of ethidium bromide to detect the produced DNAunder UV light. The respective DNA bands thus detected are excised witha razor blade.

[0909] b) Second Step PCR

[0910] LM6-DNA in which above described LM6-F 1-DNA, LM6-F2-DNA, andLM6-F3-DNA fragments are fused is prepared under the followingconditions.

[0911] Composition of the reaction solution:

[0912] Gel fragment of LM6-F1-DNA prepared in the first step PCR,

[0913] Gel fragment of LM6-F2-DNA prepared in the first step PCR,

[0914] Gel fragment of LM6-F3-DNA prepared in the first step PCR,

[0915] oligonucleotide primer 7AL1P, 50 pmol

[0916] oligonucleotide primer 7ALCN, 50 pmol

[0917] dNTPs cocktail, 5.0 μl

[0918] 10×PCR buffer, 5.0 μl

[0919] ampliTaq DNA polymerase, 2.5 units

[0920] The reaction solution having the above composition is adjusted toa final volume of 50 μl by adding redistilled water and used in PCR.

[0921] PCR thermal conditions: Heating at 94° C. for 2 minutes, afterwhich a thermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and72° C. for 2 minutes, repeated 30 times, followed by heating at 72° C.for 10 minutes.

[0922] The thus prepared LM6-DNA fragment is inserted into plasmidpCR3.1 DNA using Eukaryotic TA cloning Kit (Invitrogen) following themanufacturer's protocol and introduced into the competent E. coliTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 are confirmed by the dideoxymethod using a DNA analyzer.

[0923] The resulting plasmids are designated pCR3.1/LM6-1-16 (theplasmid carrying cDNA encoding the light chain variable region of mouseTRA-8 and a human Ig light chain constant region).

[0924] The obtained plasmid pCR3.1/LM6-1-16 containing LM6-DNA fragmentis digested with the restriction enzymes Hind III and EcoR I.

[0925] One μg of cloning plasmid pHSG399 DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pHSG399 DNA and LM6-DNA fragment,that had been digested with the restriction enzymes Hind III and EcoR I,are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM6-DNA fragment is selected by 1%agarose gel electrophoresis.

[0926] As a result of the above procedure, plasmid pHSG/M6-1-4-1carrying a fusion fragment of the variable region of the mouse TRA-8light chain and the constant region of human IgK chain is obtained. Thetransformant E coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M6-1-4-1 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7566.

[0927] 3) Construction of Plasmid pSRILM6-1-4-6(Expression Plasmid forChimera Type LM6 TRA-8 Light Chain)

[0928] The obtained plasmid pHSG/LM6-1-4-1 carrying a fusion fragment ofthe variable region of the mouse TRA-8 light chain and the constantregion of human IgK chain is digested with the restriction enzymes HindIII and EcoR I.

[0929] One μg of cloning plasmid pSRPDHH DNA is digested with therestriction enzymes Hind III and EcoR I, and then dephosphorylated withCIP. The resulting dephosphorylated pSRPDHH DNA and HindIII-EcoRI DNAfragment obtained from pHSG/LM6-1-4-1 are ligated using DNA Ligation KitVersion 2.0 (Takara Syuzo, Co. Ltd.). Then, E. coli DH5α is transformedwith the ligated DNA and spread onto LB agar. The transformants obtainedare cultured in liquid LB medium containing 100 μg/ml ampicillin, andplasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The insertion and orientation of the desired DNAfragment in the vector is confirmed by DNA sequencing using a genesequence analyzer.

[0930] The resulting expression plasmid carrying cDNA encoding the lightchain of TRA-8 (chimera type) is designated pSR/LM6-1-4-6.

[0931] (5) Production of Several Types-Humanized or Chimeric TRA-8Antibody

[0932] Transfection of COS-7 cells is conducted by FUGENE6 transfectionreagent methods (Boehringer Mannheim Biochemica) according to theinstruction manual provided with the kit.

[0933] COS-7 cells (American Type Culture Collection No. CRL-1651) aregrown to semi-confluent (3×10⁶ cells/dish) in a culture dish (culturearea: 57 cm²; Sumitomo Bakelite) containing Dulbecco's Modified Eaglemedium (hereinafter referred to as “D-MED”; Gibco BRL) supplemented with10% fetal calf serum (hereinafter abbreviated as “FCS”; Moregate).

[0934] In the meantime, 10 μg/dish (total 5 dishes) of the humanized DR5heavy chain expression plasmid DNA (pHA15-1) and 10 μg/dish of thehumanized DR5 light chain expression plasmid DNA prepared by thealkaline-SDS method and cesium chloride density gradient centrifugationare mixed, and then precipitated with ethanol, followed by suspending in5 μl/dish of dH₂O.

[0935] After 15 μl/dish of FUGENE6 Transfection regent is mixed with 180μl/dish D-MEM without FCS, this FUGENE solution (185 μl/dish) is mixedwith 5 μl/dish DNA solution containing 10 μg/dish of the humanized DR5heavy chain expression plasmid DNA and 10 μg/dish of the humanized DR5light chain expression plasmid DNA. After 15 minutes incubation at roomtemperature, the obtained plasmid suspension (200 μl) is added to thepreviously prepared COS-7 plates. After incubating in 5% CO₂ at 37° C.for 24 hours, the culture medium is changed with D-MEM without FCS.After incubating in 5% CO₂ at 37° C. for 72 hours, the culturesupernatant is recovered to purify the expression products in thesupernatant fluids. By the method as described above, COS-7 cells aretransfected with each of the following plasmid combinations:

[0936] (A): cotransfection of pHA15-1 and pSR/LM1-2 (H1L1)

[0937] (B): cotransfection of pHB14-1 and pSR/M2-1 (H2L2)

[0938] (C): cotransfection of pHB14-1 and pSR/LM3-3-44-10 (H2L3)

[0939] (D): cotransfection of pHB14-1 and pSR/LM4-5-3-3 (H2L4)

[0940] (E): cotransfection of pHC10-3 and pSR/M2-1 (H3L2)

[0941] (F): cotransfection of pHC10-3 and pSR/LM3-3-44-10 (H3L3)

[0942] (G): cotransfection of pHC10-3 and pSR/LM4-5-3-3 (H3L4)

[0943] (H): cotransfection of pHD21-1 and pSR/LM5-3-27-1 (H4L5)

[0944] (I): cotransfection of pM11-1 and pSR/LM6-1-4-6 (Chimera)

[0945] The culture is then centrifuged (3,500 r.p.m., 15 minutes) andcollected the supernatant. The supernatant is filtrated with 0.45 μmfilter (ADVANTEC TOYO DISMIC-25cs, Cat # 25CS045 AS). The purificationof IgG from the filtrates are achieved using Protein G-POROS affinitychromatography (Applied Biosystems) under the following conditions:

[0946] HPLC system: BioCAD 700E (Applied Biosystems)

[0947] column: ProteinG-ID sensor cartridge

[0948] (column size: 2.1 mmID×30 mm LD, bed volume: 0.1 ml; AppliedBiosystems)

[0949] elution buffer: 0.1 M Glycine-HCl (pH 2.5)

[0950] neutralization buffer: 1 M Tris-HCl (pH 8.5)

[0951] detection: 280 nm

[0952] flow rate: 1 ml/min

[0953] fraction size: 0.5 ml/0.5 min

[0954] fraction tube: 1.5 ml polypropylene microtube

[0955] temperature: 4° C.

[0956] After all the filtrates are applied to column, 50 ml of PBS(Sigma, Cat # 1000-3) is used to wash column. When the elution buffer isapplied, fraction collector started. Each fraction microtube previouslycontained 55 μl of 1 M NaCl, 110 μl of neutralization buffer and 74 μlof 2 mg/ml bovine serum albumin (Sigma, Cat # A-7030) in PBS. Thefractions from No. 7 through No. 8 are collected.

[0957] Verification of the expression of the humanized antibodies andquantitative assay of the expression products in the culture supernatantfluids prepared is performed by ELISA with an antibody againstanti-human IgG.

[0958] To each well of a 96-well plate (MaxiSorp, Nunc), 100 ill of goatanti-human IgG Fe specific polyclonal antibody (Kappel) dissolved at thefinal concentration of 0.5 μg/ml in adsorption buffer (0.05 M sodiumhydrogencarbonate, 0.02% sodium azide, pH 9.6) is added and the plate isincubated at 37° C. for 2 hours to cause adsorption of the antibody.Then, the plate is washed with 350 μl of PBS-T five times. To the wellsafter washing, the culture supernatant diluted with D-MEM containing 10%FCS is added and incubated at 37° C. for 2 hours. After washing againwith PBS-T, 100 ul of alkaline phosphatase-labeled goat anti-human IgGFe specific polyclonal antibody (Jackson Immuno Research Lab.) diluted10,000-fold with PBS-T is added to each well and incubated at 37° C. for2 hours. After washing again with PBS-T, a substrate solution ofp-nitrophenyl phosphate obtained from Alkaline Phosphatase Substrate kit(Bio Rad) is added according to the instruction manual provided with thekit. After incubating at 37° C. for 0.5 to 1 hour, the absorbance at 405nm is measured. In the present experiments, human plasma immunoglobulinG subclass 1 (IgG1) (Biopure AG) diluted with D-MEM containing 10% FCSto certain concentrations is used as concentration reference samples ofthe humanized DR5 antibodies contained in the culture supernatantfluids.

[0959] As a result, the expression and purified products in the culturesupernatant are detected specifically with the anti-human IgG antibody.The final concentration of human IgG antibody is 44.03 μg/ml (H1L1),39.8 μg/ml (H2L2), 26.7 μg/ml (H2L3), 41.0 μg/ml (H2L4), 39.3 μg/ml(H3L2), 24.7 μg/ml (H3L3), 21.5 μg/ml (H3L4), 16.7 μg/ml (H4L5) and 18.3μg/ml (chimera), respectively.

[0960] (6) Apoptosis-Inducing Activity of Several Types HumanizedAntibody or Chimeric Antibody

[0961] Jurkat cells (ATCC No. TIB-152), are used to examine theapoptosis-inducing activity of the purified humanized TRA-8 antibody.

[0962] Jurkat cells cultured in RPMI1640 medium with 10% FCS (Gibco BRL)at 37° C. for 3 days in the presence of 5% CO₂ are dispensed into eachwell of a 96-well microplate (Sumitomo Bakelite) at 50 μl per well. Thehumanized TRA-8 prepared in this Example 26 are adjusted to have theconcentration of the final product of interest of 100 ng/ml withRPMI1640 medium containing 10% FCS by estimating their concentrations inthe fluids according to the method described in Example 26. Each of thesolutions of the expression products thus adjusted to 100 ng/ml is usedto produce serial dilutions by repeating serial 2-fold dilution withRPMI1640 containing 10% FCS. Each of the diluted humanized TRA-8solution (H1L1, H2L2, H2L3, H2L4, H3L3, H3L4 or H4L5) is added to eachwell at 50 μl per well. After reacting at 37° C. for 12 hours, 50 μl of25 μM PMS containing 1 mg/ml XTT is added (final concentrations of 250μg/ml for XTT and 5 μM for PMS). After incubating for 3 hours, theabsorbance at 450 nm of each well is measured to calculate the cellviability by using the reduction ability of mitochondria as the index.

[0963] The viability of the cells in each well is calculated accordingto the following formula:

Viability (%)=100×(a−b)/(c−b)

[0964] wherein “a” is the measurement of a test well, “b” is themeasurement of a well with no cells, and “c” is the measurement of awell with no antibody added.

[0965] As a result, the tested humanized antibodies are demonstrated toinduce apoptosis in cells of T lymphoma cell line expressing human DR5antigen.

[0966] Furthermore, the apoptosis-inducing activity of humanized TRA-8to PC-3 is examined by adding taxol according to the method described inExample 25.

[0967] Human prostate cancer cell line PC-3 (ATCC No. CRL-1435) isobtained from American Tissue Culture Collection (ATCC) and maintainedin F-12K Nutrient Mixture (21127-022, Gibco BRL) containing 10% fetalbovine serum (FBS, Hyclone), 1% L-Glutamine-200 mM (25030-149, GibcoBRL) and 0.5% Penicillin Streptomycin Solution (P-7539, Sigma). RPMI1640medium (MED-008, IWAKI) supplemented with 10% FBS and 0.5% PenicillinStreptomycin Solution is used in the following experiment. Exponentiallygrowing PC-3 cells are collected by trypsinization and washed twice withfresh medium. The cells are then counted, resuspended in fresh medium ata density of 5×10⁴ cells/ml and distributed in triplicate intoflat-bottomed 96 well plates (3598, Coming-Coster) in a total volume of100 μl/well one day before the start of the experiment. A representativeanti-cancer drug, Paclitaxel (169-18611, Wako) dissolved indimethylsulfoxide (10 mg/ml) is diluted in fresh medium and then addedto the 96-well plates containing the cells at 50 μl/well. The finalconcentrations of dimethylsulfoxide are less than 0.1%. After incubationfor 24 hr at 37° C. in 5% CO₂ atmosphere, humanized TRA-8 antibody(H1L1, H2L2, H2L3, H2L4, H3L2, H3L3, H3L4 or H4L5) diluted in freshmedium is added to the wells. After incubation for a further 24 hr, 50μl of Minimum Essential Medium (11095-098, Gibco BRL) containing 1 mg/mlof XTT and 25 mM of PMS is added to the wells and the plates areincubated for 6 hr. OD450 is then measured by ARVO HTS 1420 MultilabelCounter (Wallac Berthold) and the cell viability is calculated asfollows.

Cell viability (%)=(OD450 for the well containing cells treated withTaxol and humanized TRA-8 (agent(s))−OD450 for the well containingneither cells nor agent)×100/(OD450 for the well containing cells withno agent−OD450 for the well containing neither cells nor agent)

[0968] As a result, the tested humanized antibodies are demonstrated toinduce apoptosis in human prostate cancer cells expressing human DR5antigen.

EXAMPLE 27 Production of DR4 Antibody

[0969] A fusion protein containing the extracellular domain of human DR4(a.a. 1-236) and the Fc portion of human IgG1 was expressed in Cos-7cells transfected with a recombinant adenoviral vector. The fusionprotein was purified by protein A affinity column. Balb/c mice wereimmunized with the purified fusion protein as described above. Onehybridoma clone, 2E12 (IgG1, κ), with specific binding to DR4 and thecapability of inducing apoptosis of Ramos human B lymphoma cells wassubcloned three times. The binding specificity of 2E12 was determined byELISA and Western blot analysis using human DR5, DcR1 and DcR2 and IgG1fusion protein as control antigens. The binding of 2E12 to cell surfaceDR4 was determined by flow cytometry analysis of Cos-7 cells transfectedwith the full-length cDNA encoding human DR4. Apoptosis-inducingactivity was determined by incubating Ramos cells with 1 μg/ml 2E12 inthe presence of goat anti-mouse IgG1. Cell viability was determined byATPLite assay as described above.

EXAMPLE 28 Characterization of DR4 Antibody

[0970] DR4 monoclonal antibody (2E12) is specific for human DR4 as itdid not bind to other TRAIL receptors such as DR5, DcR1 and DcR2 inELISA (FIG. 19a). 2E12 recognized cell surface DR4 as demonstrated byflow cytometry analysis of DR4 transfected Cos-7 cells (FIG. 19b). 2E12was able to induce apoptosis of Ramos lymphoma cells in the presence ofsecond antibody crosslinking in a dose-dependent fashion (FIG. 19c). Invitro treatment of Ramos cells with 2E12 resulted in a time-dependentactivation of caspase 8, 9 and 3, and cleavage of PARP (FIG. 19d). Theseresults indicate that 2E12 is an agonistic anti-DR4 antibody, whichinduces apoptosis in a caspase-dependent fashion.

[0971] Using anti-DR4 (2E12), followed by PE-conjugated goat anti-mouseIgG1 antibody and flow cytometry, DR4 antibody was shown to bind tocells of a fibrosarcoma cell line (Hs 913T) and several breast cancercell lines (2LMP. MDA-MB231, and MDA-MB 453), but showed little to nobinding to a normal human skin fibroblast cell line (Malme-3).

EXAMPLE 29 Tumoricidal Activity of DR4 Antibodies

[0972] The tumoricidal activity of 2E12 was tested using breast cancertumor models. Nude mice were inoculated s. c. with the human breastcancer cell line, 2LMP. Treatment with i.p. doses of 200 μg of 2E12occurred on days 7, 10, 14, 17, 21, and 24 after tumor cell injection.Animals received i.v. adriamycin (doxorubicin) (6 mg/kg) on days 8, 12,and 16. Treatment with 2E12 and adriamycin (FIG. 20) produced greatertumor growth inhibition than either 2E12 or adriamycin alone.

[0973] The tumoricidal activity of TRA-8 in combination with 2E12 wastested using the same breast cancer tumor models. Treatment with i.p.doses of 200 μg of TRA-8 and 2E12 occurred on days 7, 10, 14, 17, 21,and 24 after tumor cell injection. Animals received i.v. adriamycin (6mg/kg) on days 8, 12, and 16. Treatment with TRA-8 plus 2E12, or TRA-8plus 2E12 and adriamycin produced 88% and 100% complete tumorregression, respectively. (FIG. 21.)

EXAMPLE 30 Tumoricidal Activity of DR4/DR5 Antibodies in Combinationwith Other Therapies

[0974] (1) Cell Lines and Reagents

[0975] The 2LMP subclone of the human breast cancer cell lineMDA-MB-231, the LCC6 subclone of MDA-MB-435, and the DY36T2 subclone ofMDA-MB-361 were obtained from Dr. Marc Lipmann (Georgetown University,Washington, D.C.) and maintained in improved MEM supplemented with 10%FBS (Hyclone, Logan, Utah). The MDA-MB-231, MDA-MB-453, MDA-MB-468,BT-474, SK-BR-3, and ZR-75-1 human breast cancer cell lines wereobtained from the American Type Culture Collection (Manassas, Va.).MDA-MB-231, MDA-MB-453, and MDA-MB-468 cells were grown in DMEMsupplemented with MEM vitamins, MEM nonessential amino acids, 1 mMsodium pyruvate and 10% FBS. BT-474 cells were grown in RPMI 1640supplemented with 10 μg/ml insulin, 4.5 g/l glucose, 10 mM HEPES, 1 mMsodium pyruvate and 10% FBS. SK-BR-3 cells were grown in McCoy's mediumwith 15% FBS. ZR-75-1 cells were grown in Ham's F12K medium with 20%FBS. All cell lines were maintained in antibiotic-free medium at 37° C.in a 5% CO₂ atmosphere and routinely screened for mycoplasmacontamination

[0976] Purified TRA-8 (IgG1) mAb was produced at UAB and also providedby Sankyo Co., Ltd. (Tokyo, Japan). Phycoerythrin-conjugated goatanti-mouse IgG1 and isotype-specific IgG1 control antibody were obtainedfrom Southern Biotechnology Associates (Birmingham, Ala.). Adriamycinand paclitaxel were purchased from Sigma Chemical Co. (St. Louis, Mo.)and were prepared as 10 mM stock solutions in distilled H20 or DMSO,respectively. For animal studies, the clinical formulation of paclitaxel(Bristol-Myers Squibb Co., Princeton, N.J.) was obtained from theUniversity of Alabama at Birmingham Hospital Pharmacy (Birmingham,Ala.). This preparation was diluted 1:5 in PBS immediately before use.

[0977] (2) Indirect Immunofluorescence and Flow Cytometry Analysis ofDR5 Expression

[0978] Cells in exponential growth phase were washed once withDulbecco's PBS (Ca2+ and Mg2+deficient) and harvested with 4 mMEDTA/0.5% KCl at 37° C. Cells were collected by centrifugation at 4° C.for 5 min at 1,000 rpm, washed once and resuspended in PBS containing 1%BSA and 0.01% sodium azide (FACS buffer) at 4° C. Cells were incubatedwith 10 μg/ml of purified TRA-8 or an isotype-specific IgG1 controlantibody for 60 min at 4° C., washed once with buffer, then incubatedwith 10 μg/ml of PE-conjugated goat anti-mouse IgG1 for 20 min at 4° C.After antibody staining, cells were washed once with FACS buffer andfixed in 1% paraformaldehyde for 15 min on ice. Samples were analyzed ona Becton Dickinson FACScan (San Jose, Calif.) and data was analyzedusing CellQuest software.

[0979] (3) Cell Viability Assays Using ATPLite

[0980] Cells were trypsinized and resuspended in complete culturemedium. One thousand cells per well were plated in optically clear96-well black plates (Costar #3904, Corning, N.Y.) and incubatedovernight at 37° C. before initiating treatments. Drugs and antibodywere diluted in culture medium immediately before use, and the finalconcentration of DMSO was always ≦0.001%. Cell viability was assessedafter 24 h exposure to TRA-8 alone. For combination treatments withcytotoxic drugs, cells were pretreated with the drug for 24 h beforeadding antibody, and incubated for an additional 24 h before assessingcell viability by measurement of cellular ATP levels using the ATPLiteluminescence based assay (Packard Instruments, Meriden, Conn.). Themanufacturer's recommended protocol was followed with the exception thatall reaction volumes (culture medium and reagents) were reduced byone-half. All samples were assayed in triplicate and are reported as themean±SE from a minimum of 3 independent experiments.

[0981] (4) TRA-8 Therapy Studies Alone or in Combination withChemotherapy or Radiation in Athymic Nude Mice Bearing Breast CancerXenografts

[0982] Athymic nude mice were injected s.c. with 3×107 2LMP cells. At 7days after tumor cell injection, 200 or 600 μg (10 or 30 mg/kg) TRA-8was administered i.p. followed by five additional injections on days 10,14, 17, 21, and 24. The growth of tumors was monitored over time. Insubsequent studies, animals bearing 2LMP s.c. tumors were injected i.p.with 200 μg of TRA-8 on days 7, 10, 14, 17, 21, and 24 alone or incombination with adriamycin (6 mg/kg i.v., days 8, 12, and 16) orpaclitaxel (20 mg/kg i.p., on days 8, 12, 16, 20, and 24). Tumor sizeand regression rates were determined. In addition, a study was carriedout with TRA-8 and adriamycin using the same regimen described above incombination with 3 Gy ⁶⁰Co irradiation of 2LMP xenografts on days 9 and17.

[0983] (5) Analysis of Apoptosis in Xenografts

[0984] Athymic nude mice injected s.c. with 3×10 2LMP cells on day 0received 100 μg TRA-8 i.p. on days 7 and 10. Groups of 2 mice eachreceived adriamycin (3 mg/kg) on days 8 and 11, paclitaxel (10 mg/kg) ondays 8 and 111, or the combination of TRA-8 and adriamycin or paclitaxelwith the same dose and schedule. One group of mice was untreated. Thexeno grafts were dissected for the study of apoptosis on day 14 aftertumor cell injection. The reason for the substantial reduction intreatment intensity compared to our standard treatment protocol was toallow adequate tumor tissue for analysis on day 14. Tunel assay forapoptosis in tumor xenografts was performed as follows. Five-micronparaffin sections of tissue were mounted on Superfrost/Plus slides andheated at 58° C. for 1 h. Tissue sections were deparaffinized in threechanges of xylene and rehydrated with one change of absolute ethanol,95% ethanol, and 70% ethanol, each in 5-min increments. Then, thesections were placed in Tris-buffered saline (0.5M Tris base, 0.15 MNaCl, 0.0002% Triton X-100, pH 7.6). Apoptotic nuclei were detectedusing an Apop Tag Peroxidase kit (Intergen, Purchase, N.Y.). ProteinaseK (20 μg/ml in distilled deionized H₂O) was added to the tissuespecimens and incubated at room temperature for 15 min. Endogenousperoxidases were quenched with an aqueous solution of 3% hydrogenperoxide for 5 min. Sections were treated with an equilibration bufferfor 30 min and then incubated with the TdT/enzyme (diluted in labelingreaction mix) for 1 h at 37° C. using parafilm covers. During thisincubation, the TdT enzyme binds the 3′-OH ends of DNA fragments andcatalyzes the addition of digoxigenin-labeled and unlabeleddeoxynucleotides. Negative controls were incubated with distilled H₂O(diluted in labeling reaction mix) instead of the TdT enzyme. A stopbuffer was added for 10 min at room temperature to terminate thelabeling reaction. An anti-digoxigenin conjugate was added to each slidefor 30 min. The chromagen 3,3′ DAB was used to visualize the labeled3′OH end of DNA fragments. The slides were then rinsed in deionizedwater and lightly counterstained with hematoxylin, dehydrated usinggraded alcohols and xylene and coverlsipped using Permount.Approximately 10 random fields were evaluated for percentage of Tunelstained and percentage of intensely stained apoptotic bodies throughoutthe tissue.

[0985] (6) Statistical Analysis

[0986] (A) Analysis of TRA-8 Interaction with Drug Cytotoxicity In Vitro

[0987] The cytotoxicity data were evaluated to assess whether thecombination cytotoxic effects were additive, less than additive(antagonistic), or greater than additive (synergistic). The doseresponse relationships for the agents alone and in combination weremodeled using a second-order response surface model with linear,quadratic and interaction terms for each of the 9 cell lines(Montgomery, D.C. Design and Analysis of Experiments, New York:Wiley,2001), as recommended by Gennings (On Testing for Drug/ChemicalInteractions: Definitions and Inference, pp. 457-468, 2000). Asignificant interaction term was classed as either synergistic orantagonistic depending on whether the interaction term was negative withmore than additive cytotoxicity, or positive with less than additivecytotoxicity. If the interaction term was not significant, then therelationship between TRA-8 and adriamycin or TRA-8 and paclitaxel wouldbe considered additive, provided the additive terms were significant.

[0988] (B) Analysis of TRA-8, Chemotherapy, Radiation and CombinationTherapy of Individual Animal Experiments

[0989] Data from 6 independent experiments were analyzed by individualexperiment. Treatment combinations were compared with respect to in vivoanti-tumor efficacy, i.e., inhibition of tumor growth, which wasmeasured as three endpoints; extension of tumor doubling times,percentage of tumor regressions, and growth rates over time. The actualnumber of days at which the tumor doubled in surface area (product oftwo diameters) relative to baseline on day 7 after tumor cell injectionwas used in the doubling time analysis. The nonparametric Kruskal-Wallistest was used for median tumor doubling time comparisons betweentreatments. Fisher's exact test was used to compare the proportions oftumor regressions and relapse-free regressions across treatment groups.To determine if any combination therapy produced significant synergisticinhibition of tumor growth, i.e., more than additive, the growth curvesfrom the serial area measurements were compared using a linear mixedmodel approach over the first 3 weeks after start of therapy (Lindsey,J. K. Models for Repeated Measurements, pp. 100-142, Oxford, 1993). Totest for synergistic effects of the combination therapies, aninteraction term was included in the model. If the interaction term wassignificant and the effect was inhibition of growth at a rate greaterthan additive then the interaction was considered synergistic.

[0990] (C) Aggregate Analysis of Therapy Effects

[0991] A total of 166 animals, 10 treatment groups, and 6 independentexperiments were included in the aggregate analysis. Treatmentcombinations were compared with respect to in vivo anti-tumor efficacy.The median tumor doubling times were analyzed using the Kruskal-Wallistest and Fisher's exact test was used to compare the proportions oftumor regressions and relapse-free regressions across treatment groups.

[0992] All statistical analyses were conducted using SAS® (Sas/StatUser's Guide, SAS OnlineDoc, Version 8, Cary N.C.: SAS Institute Inc.,1999).

[0993] (6) DR5 Expression and TRA-8 Induced Cytotoxicity in BreastCancer Cell Lines

[0994] As illustrated in FIG. 22A, all nine breast cancer cell lineswere DR5 positive with varying degrees of expression from stronglypositive (LCC6 and MDA-MB-453) to weakly positive (MDA-MB-468 andSK-BR-3). FIG. 22B illustrates the TRA-8 induced cytotoxicity of thenine cell lines. Four cell lines were sensitive to TRA-8 inducedcytotoxicity with IC₅₀ concentrations of 17 to 299 ng/ml (LCC6, 2LMP,MDA-MB-231, MDA-MB-468), while others were quite resistant (DY36T2,BT-474, MDA-MB-453). There was not a good correlation of DR5 expressionand degree of TRA-8 induced cytotoxicity as illustrated by cell linesMDA-MB-453 and MDA-MB-468.

[0995] TRA-8 effects on chemotherapy-induced cytotoxicity were thenexamined with adriamycin (FIG. 23A) and paclitaxel (FIG. 23B). Ananalysis to test for interaction between antibody and drug effects issummarized in Table 5. There were no significant synergisticinteractions between TRA-8 and paclitaxel, with most of the interactionsbeing additive. Four of nine cell lines fulfilled criteria for asynergistic interaction between TRA-8 and adriamycin. The cell line 2LMPdemonstrated good sensitivity to TRA-8, as well as sensitivity to eitheradriamycin or paclitaxel. This cell line was chosen to explore in vivoefficacy of antibody and/or drugs. TABLE 5 In Vitro Interaction Effectsfor Combination Treatments TRA-8 + Adriamycin TRA-8 + Paclitaxel CellLine Interaction p-value^(a) Interaction p-value^(a) LCC6 Synergistic<0.001 Additive 0.624 MDA-MB-453 Synergistic <0.001 No response^(c)0.615 2LMP Additive 0.153 Additive 0.937 MDA-MB-231 Additive 0.663Additive 0.064 BT-474 Synergistic <0.001 ND^(b) 0.992 ZR-75-1Synergistic 0.013 Additive 0.172 DY36T2 ND^(b) 0.808 ND^(b) 0.798MDA-MB-468 Additive 0.184 Additive 0.724 SK-BR-3 Additive 0.361 Noresponse^(c) 0.871

[0996] (7) In Vivo Anti-Tumor Effects of TRA-8 Alone or in Combinationwith Chemotherapy and/or Radiation

[0997] TRA-8 at doses of 200 μg and 600 μg twice a week for 6 dosesproduced a similar inhibition of tumor growth for well-established 2LMPs.c. tumors (FIG. 24). In three additional independent experiments, the200 μg dose/schedule produced statistically significant inhibition oftumor growth (p<0.004, Kruskal-Wallis test on tumor doubling times)compared to untreated controls and this dose and schedule was selectedfor further studies. FIG. 25 illustrates the effects of TRA-8,adriamycin, or a combination of TRA-8 and adriamycin on anti-tumorefficacy. As compared to untreated controls, therapy with TRA-8 alone orTRA-8 plus adriamycin produced significant inhibition of tumor growth(p=0.002 Kruskal-Wallis test), while adriamycin did not differ fromcontrols. The combination of TRA-8 plus adriamycin produced greatergrowth inhibition than either agent alone (p=0.002), as well assignificantly more complete regressions of tumor (four) than eitheragent alone where no complete regressions were seen (p<0.001, Fisherexact test). In vivo TRA-8 and adriamycin synergism was evaluated usingan early growth curve analysis. The interaction term was significant(p<0.001) and synergistic. The synergistic interaction was corroboratedin a second independent experiment.

[0998] The effects of TRA-8 and paclitaxel were studied in this samemodel with similar observations (FIG. 26). As compared to untreatedcontrols, TRA-8 and the TRA-8 plus paclitaxel produced significantinhibition of tumor growth (p<0.001, Kruskal-Wallis test). Tumor growthin animals treated with TRA-8 plus paclitaxel was significantlydifferent than paclitaxel alone (p=0.008) and produced 3/8 completeregressions as compared to none for either agent alone. Analysis of theearly tumor growth curves demonstrated that synergistic effect wasnearly significant (p=0.063) while additive effects were significant(p<0.001).

[0999] Finally, the effects of TRA-8, adriamycin, and ⁶⁰Co radiationwere analyzed as single agents and in various combinations asillustrated in FIG. 27. There were significant differences overall withrespect to tumor doubling times (p<0.001) and multiple comparisonsindicated that the triple therapy with TRA-8, adriamycin, and ⁶⁰Coproduced tumor growth inhibition that was significantly different thanall other treated groups, while both dual therapy groups (adriamycinplus TRA-8 or ⁶⁰Co plus TRA-8) were different than either single agentgroup (p<0.001). The ⁶⁰Co animals treated with radiation alone did notdiffer from untreated controls (p=0.926). All two-way treatmentcombinations had significant synergistic effects (p<0.001). Completeregressions were seen in 6/8 animals receiving triple therapy and 4animals did not have tumor recurrence over 180 days of follow-up.

[1000] (8) Aggregate Analysis of Therapy Effects

[1001] The in vivo anti-tumor studies were comprised of 166 animals, andthe tumor doubling times and frequency of complete tumor regression forall animals in each treatment group were analyzed (Table 6). ANOVAanalysis for mean tumor doubling times indicated significant differencesamong treatment groups (p<0.001), with multiple comparisons yieldingthat TRA-8+paclitaxel, TRA-8+adriamycin, and TRA-8+adriamycin+⁶⁰Co hadsignificantly longer mean tumor doubling times than any treatment grouplacking TRA-8. The addition of TRA-8 to any treatment modality produceda longer tumor doubling time than that modality alone. Similarly,Kruskal-Wallis test on median time to tumor doubling yielded that themedians were significantly different over-all (p<0.001). Pair-wisecomparisons using Wilcoxin signed-rank test yielded similar patterns formedian time to tumor doubling as the ANOVA multiple comparisons. Thisanalysis underestimates the growth inhibition produced by the mosteffective treatments in that groups that did not reach a doubling oftumor by the end of the experiment were assigned the experimenttermination day. Table 6 also provides the frequency of completeregression of tumor and the frequency of persistence of that regressionto the end of the experiment. There were no complete regressions oftumor seen in animals treated with either chemotherapy regimen orradiation attesting to the well-established tumor growth and tumoraggressiveness. From Fisher's exact test, there were significantdifferences in the frequency of tumor complete regressions betweentreatment groups (p<0.001). Thirty of 166 animals achieved completeregression, and 28 of these received TRA-8 alone or in combination withother modalities. Complete regression occurred in 1/42 control animals:1/54 animals receiving chemotherapy, radiation, or a combination; and28/68 of TRA-8 alone or TRA-8 combination regimens. The TRA-8 treatedgroups had a significantly (p<0.001) greater frequency of completeregression. Similarly, 14/68 animals receiving TRA-8 or TRA-8combinations did not have tumor re-growth compared to 1/42 controls and0/52 animals treated with chemotherapy and/or radiation. Therelapse-free regressions had observation periods of 99 to 171 days(146±24 days). TABLE 6 Aggregate Results of Doubling Time and CompleteRegression of 2LMP Tumors Complete Regressions # of Tumor Doubling Time(days) Total No relapse Mean Observat Treatment Animals (mean/median)(%) (%) Period (days Untreated Controls 44 (42)^(a) 12/8  1 (2%)  1(2%)  177 ⁶⁰Co  8 (7) 14/10 0 0 186 Adriamycin 31 (28) 17/18 0 0 197Paclitaxel  7 (5) 25/20 0 0 — Adriamycin + ⁶⁰ Co  8 (8) 39/36 1 (13%) 0197 TRA-8 30 (26) 47/23 6 (20%) 5 (17%) 159 TRA-8 + ⁶⁰Co  8 (8) 65/50 3(38%) 1 (13%) 186 TRA-8 + Paclitaxel  8 (8) 71/62 3 (38%) 1 (13%) 148TRA-8 + Adriamycin 14 (12) 81/64 10 (71%)  3 (21%) 185 TRA-8 +  8(6) >140/179  6 (75%) 4 (50%) 192 Adriamycin + ⁶⁰Co

[1002] (9) Apoptosis in Treated Tumors

[1003] The induction of apoptosis in 2LMP xenografts following treatmentwith TRA-8, adriamycin, paclitaxel, TRA-8+adriamycin, andTRA-8+paclitaxel was assessed using the TUNEL technique. In untreatedanimals, tumors had 4% stained cells (1% intense), while treatment withadriamycin or paclitaxel had 8% (6% intense) and 7% (2% intense) stainedcells. Animals treated with TRA-8 alone had striking apoptosis with 25%(15% intense) stained cells. TRA-8 plus adriamycin had 28% (22% intense)and TRA-8 plus paclitaxel had 26% (12% intense) stained cells.

[1004] Any patents or publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[1005] The present invention is not limited in scope by theabove-referenced deposit or the embodiments disclosed in the exampleswhich are intended as illustrations of a few aspects of the inventionand any embodiments which are functionally equivalent are within thescope of this invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art and are intended to fall within the scope ofthe appended claims.

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0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 102 <210> SEQ ID NO 1<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 1 gacgatgcccgatctacttt aaggg 25 <210> SEQ ID NO 2 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 2 ccactgggtg atgttggatg gg 22 <210> SEQ ID NO3 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 3 ggatccgtggacacattcga tgtc 24 <210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 4 Glu Val Met Leu Val Glu Ser Gly Gly Gly LeuVal Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu 20 <210> SEQ ID NO 5 <211>LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 5 Asp Ile Val Met Thr GlnSer His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser 20<210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 6cagcactgaa cacggacccc 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 7 aaaggtaatt tattgagaag U20 <210> SEQ ID NO 8<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 8 cctcaccatgaacttcgggc U20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 9 ctgttgtatg cacatgagac U20 <210> SEQ ID NO 10 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 10 gaagtgatgc tggtggagtc U20 <210>SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 11agtgtgaagt gatgctggtg U20 <210> SEQ ID NO 12 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 12 tttaccagga gagtgggaga g U21 <210> SEQ ID NO13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 13 tgcagagacagtgaccagag U20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 14 tgttcaggac cagcatgggc U20 <210> SEQ ID NO 15 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 15 aagacatttt ggattctaac U20 <210>SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 16tatcatgaag tctttgtatg U20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 17 gatggagaca cattctcagg U20 <210> SEQ ID NO18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 18 gacattgtgatgacccagtc U20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 19 ttaacactca ttcctgttga U20 <210> SEQ ID NO 20 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 20 gactgggtca tcacaatgtc 20 <210>SEQ ID NO 21 <211> LENGTH: 1386 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 21atgaacttcg ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60gtgatgctgg tggagtctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagc tatgtaatgt cttgggttcg ccagactccg 180gagaagaggc tggagtgggt cgcaaccatt agtagtggtg gtagttacac ctactatcca 240gacagtgtga aggggcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaggtc tgaggacacg gccatgtatt actgtgcaag acggggggac 360tctatgatta cgacggacta ctggggccaa ggcaccactc tcacagtctc ctcagccaaa 420acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 480gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 540tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 600actctgagca gctcagtgac tgtcccctcc agcacctggc ccagcgagac cgtcacctgc 660aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 720ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 780aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 840atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 900acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa 960cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 1020gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1080ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1140acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg 1200cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc 1260gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1320tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1380ggtaaa 1386 <210> SEQ ID NO 22 <211> LENGTH: 705 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 22 atgaagtctt tgtatgtgtt agtgtataca cattatctgt ttctgtttgcaggtgttgaa 60 ggagacattg tgatgaccca gtctcacaaa ttcatgtcca catcagtaggagacagggtc 120 agcatcacct gcaaggccag tcaggatgtg ggtactgctg tagcctggtatcaacagaaa 180 ccagggcaat ctcctaaact actgatttac tgggcatcca cccggcacactggagtccct 240 gatcgcttca caggcagtgg atctgggaca gatttcactc tcaccattagcaatgtgcag 300 tctgaagact tggcagatta tttctgtcag caatatagca gctatcggacgttcggtgga 360 ggcaccaagc tggaaatcaa acgggctgat gctgcaccaa ctgtatccatcttcccacca 420 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaacaacttctac 480 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaatggcgtcctg 540 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcagcaccctcacg 600 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccactcacaagaca 660 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttaa 705<210> SEQ ID NO 23 <211> LENGTH: 462 <212> TYPE: PRT <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 23Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 1015 Val Gln Cys Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 2530 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 4045 Ser Ser Tyr Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 5560 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro 65 7075 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 8590 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met100 105 110 Tyr Tyr Cys Ala Arg Arg Gly Asp Ser Met Ile Thr Thr Asp TyrTrp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr ThrPro Pro 130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln ThrAsn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr PhePro Glu Pro Val Thr 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser SerGly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr ThrLeu Ser Ser Ser Val Thr Val 195 200 205 Pro Ser Ser Thr Trp Pro Ser GluThr Val Thr Cys Asn Val Ala His 210 215 220 Pro Ala Ser Ser Thr Lys ValAsp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro CysIle Cys Thr Val Pro Glu Val Ser Ser Val Phe 245 250 255 Ile Phe Pro ProLys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 260 265 270 Lys Val ThrCys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 275 280 285 Gln PheSer Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 290 295 300 GlnPro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 305 310 315320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 325330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile ProPro 355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr CysMet Ile 370 375 380 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp GlnTrp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln ProIle Met Asp Thr Asp 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu AsnVal Gln Lys Ser Asn Trp 420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys SerVal Leu His Glu Gly Leu His 435 440 445 Asn His His Thr Glu Lys Ser LeuSer His Ser Pro Gly Lys 450 455 460 <210> SEQ ID NO 24 <211> LENGTH: 234<212> TYPE: PRT <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 24 Met Lys Ser Leu Tyr Val Leu Val Tyr Thr HisTyr Leu Phe Leu Phe 1 5 10 15 Ala Gly Val Glu Gly Asp Ile Val Met ThrGln Ser His Lys Phe Met 20 25 30 Ser Thr Ser Val Gly Asp Arg Val Ser IleThr Cys Lys Ala Ser Gln 35 40 45 Asp Val Gly Thr Ala Val Ala Trp Tyr GlnGln Lys Pro Gly Gln Ser 50 55 60 Pro Lys Leu Leu Ile Tyr Trp Ala Ser ThrArg His Thr Gly Val Pro 65 70 75 80 Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Asn Val Gln Ser Glu Asp Leu AlaAsp Tyr Phe Cys Gln Gln Tyr 100 105 110 Ser Ser Tyr Arg Thr Phe Gly GlyGly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp Ala Ala Pro Thr ValSer Ile Phe Pro Pro Ser Ser Glu Gln 130 135 140 Leu Thr Ser Gly Gly AlaSer Val Val Cys Phe Leu Asn Asn Phe Tyr 145 150 155 160 Pro Lys Asp IleAsn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 165 170 175 Asn Gly ValLeu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr SerMet Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 195 200 205 HisAsn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 210 215 220Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 <210> SEQ ID NO 25 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 25 Ser Tyr Val Met Ser 1 5<210> SEQ ID NO 26 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 26Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 1015 Gly <210> SEQ ID NO 27 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 27 Arg Gly Asp Ser Met Ile Thr Thr Asp Tyr 1 5 10 <210> SEQ IDNO 28 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 28 LysAla Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> SEQ ID NO 29 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 29 Trp Ala Ser Thr Arg HisThr 1 5 <210> SEQ ID NO 30 <211> LENGTH: 8 <212> TYPE: PRT <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 30 Gln Gln Tyr Ser Ser Tyr Arg Thr 1 5 <210> SEQ ID NO 31<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 31 Glu Val Met LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met SerTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr IleSer Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly ArgPhe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala ArgArg Gly Asp Ser Met Ile Thr Thr Asp Tyr Trp Gly Gln Gly 100 105 110 ThrLeu Val Thr Val Ser Ser 115 <210> SEQ ID NO 32 <211> LENGTH: 80 <212>TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 32 ttggataagc ttggcttgac ctcaccatgg gatggagctgtatcatcctc ttcttggtag 60 caacagctac aggtgtccac 80 <210> SEQ ID NO 33<211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 33 tctgaagtaatgctggtgga gtctggggga ggcttagtac agcctggagg gtccctgaga 60 ctctcctgtgcagcctctgg 80 <210> SEQ ID NO 34 <211> LENGTH: 80 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 34 attcactttc agtagttatg taatgtcttg ggttcggcag gcaccagggaagggtctgga 60 gtgggttgca accattagta 80 <210> SEQ ID NO 35 <211> LENGTH:80 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 35 gtggtggtag ttacacctac tatccagacagtgtgaaggg ccgattcacc atctccagag 60 acaatgccaa gaacaccctg 80 <210> SEQID NO 36 <211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 36tatctgcaaa tgaacagtct gagagcagag gacacggctg tttattactg tgcaagaagg 60ggtgactcta tgattacgac 80 <210> SEQ ID NO 37 <211> LENGTH: 64 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 37 ggactactgg ggccaaggga ccctggtcac agtctcctcagcctccacca agggcccatc 60 ggtc 64 <210> SEQ ID NO 38 <211> LENGTH: 60<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 38 ctaccaagaa gaggatgata cagctccatc ccatggtgaggtcaagccaa gcttatccaa 60 <210> SEQ ID NO 39 <211> LENGTH: 80 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 39 tctcagggac cctccaggct gtactaagcc tcccccagactccaccagca ttacttcaga 60 gtggacacct gtagctgttg 80 <210> SEQ ID NO 40<211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 40 tccagacccttccctggtgc ctgccgaacc caagacatta cataactact gaaagtgaat 60 ccagaggctgcacaggagag 80 <210> SEQ ID NO 41 <211> LENGTH: 80 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 41 ctctggagat ggtgaatcgg cccttcacac tgtctggata gtaggtgtaactaccaccac 60 tactaatggt tgcaacccac 80 <210> SEQ ID NO 42 <211> LENGTH:80 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 42 ccttcttgca cagtaataaa cagccgtgtcctctgctctc agactgttca tttgcagata 60 cagggtgttc ttggcattgt 80 <210> SEQID NO 43 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 43gaccgatggg cccttggtgg aggctgagga gactgtgacc agggtccctt ggccccagta 60gtccgtcgta atcatagagt cacc 84 <210> SEQ ID NO 44 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 44 ttggataagc ttggcttgac 20 <210> SEQ ID NO 45<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 45 gaccgatgggcccttggtgg a 21 <210> SEQ ID NO 46 <211> LENGTH: 213 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 46 Asp Ile Val Met Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala SerGln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly ValPro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys GlnGln Tyr Ser Ser Tyr Arg Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu IleLys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro SerAsp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu LeuAsn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val AspAsn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr GluGln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 AsnArg Gly Glu Cys 210 <210> SEQ ID NO 47 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 47 cccaagctta agaagcatcc tctcatctag ttct 34<210> SEQ ID NO 48 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 48cccgaattct tactaacact ctcccctgtt gaagctcttt gtgac 45 <210> SEQ ID NO 49<211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 49 gtcccccacagatgcagaca aagaacttgg agattgggtc atcacaatgt caccagtgga 60 <210> SEQ IDNO 50 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 50ccaagttctt tgtctgcatc agtaggagac agggtcacca tcacctgc 48 <210> SEQ ID NO51 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 51 agtgtgccgggtggatgccc agtaaatcag tagtttagga gctttccctg gtttctg 57 <210> SEQ ID NO52 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 52 tgggcatccacccggcacac tggggtccca agcaggttta gtggcagt 48 <210> SEQ ID NO 53 <211>LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 53 ataactacta tattgctgacagtaataggt tgcaaaatcc tccggctgca gactagagat 60 ggt 63 <210> SEQ ID NO 54<211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 54 cagcaatatagcagctatcg gacgttcggt caaggcacca aggtggaaat caaacggact 60 gtg 63 <210>SEQ ID NO 55 <211> LENGTH: 711 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 55atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 60gacattgtga tgacccaatc tccaagttct ttgtctgcat ctgtggggga cagggtcacc 120atcacctgca aggccagtca ggatgtgggt actgctgtag cctggtatca acagaaacca 180gggaaagctc ctaaactact gatttactgg gcatccaccc ggcacactgg ggtcccaagc 240aggtttagtg gcagtgggtc tgggacagac ttcaccctca ccatctctag tctgcagccg 300gaggattttg caacctatta ctgtcagcaa tatagtagtt atcggacgtt cggtcaaggc 360accaaggtgg aaatcaaacg gactgtggct gcaccatctg tcttcatctt cccgccatct 420gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660agctcgcccg tcacaaagag cttcaacagg ggagagtgtt agtaagaatt c 711 <210> SEQID NO 56 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 56 GluVal Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 7580 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095 Ala Arg Arg Gly Asp Ser Met Ile Thr Thr Asp Tyr Trp Gly Gln Gly 100105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 57 <211> LENGTH:80 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 57 tctgaagtac agctggtgga gtctgggggaggcttagtac agcctggagg gtccctgaga 60 ctctcctgtg cagcctctgg 80 <210> SEQID NO 58 <211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 58tctcagggac cctccaggct gtactaagcc tcccccagac tccaccagct gtacttcaga 60gtggacacct gtagctgttg 80 <210> SEQ ID NO 59 <211> LENGTH: 119 <212>TYPE: PRT <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 59 Glu Val Met Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr ThrTyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnAla Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Asp Ser Met Ile ThrThr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115<210> SEQ ID NO 60 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:artificial sequence <220> FEATURE: 223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 60Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 4045 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 5560 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 7075 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 Ala Arg Arg Gly Asp Ser Met Ile Thr Thr Asp Tyr Trp Gly Gln Gly100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 61 <211>LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 61 Glu Val Met Leu Val GluSer Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp ValArg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser SerGly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe ThrIle Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met SerSer Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg GlyAsp Ser Met Ile Thr Thr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr LeuThr Val Ser Ser 115 <210> SEQ ID NO 62 <211> LENGTH: 80 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 62 tatctgcaaa tgagcagtct gagagcagag gacacggctgtttattactg tgcaagaagg 60 ggtgactcta tgattacgac 80 <210> SEQ ID NO 63<211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 63 ccttcttgcacagtaataaa cagccgtgtc ctctgctctc agactgttca tttgcagata 60 cagggtgttcttggcattgt 80 <210> SEQ ID NO 64 <211> LENGTH: 80 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 64 attcactttc agtagttatg taatgtcttg ggttcggcag actccagagaagaggctgga 60 gtgggttgca accattagta 80 <210> SEQ ID NO 65 <211> LENGTH:80 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: /Note =Synthetic Construct <400> SEQUENCE: 65 tccagcctct tctctggagt ctgccgaacccaagacatta cataactact gaaagtgaat 60 ccagaggctg cacaggagag 80 <210> SEQID NO 66 <211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 66tctgaagtaa tgctggtgga gtctggggga ggcttagtaa agcctggagg gtccctgaaa 60ctctcctgtg cagcctctgg 80 <210> SEQ ID NO 67 <211> LENGTH: 80 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 67 tatctgcaaa tgagcagtct gagatctgag gacacggctatgtattactg tgcaagaagg 60 ggtgactcta tgattacgac 80 <210> SEQ ID NO 68<211> LENGTH: 64 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 68 ggactactggggccaaggga ccactctcac agtctcctca gcctccacca agggcccatc 60 ggtc 64 <210>SEQ ID NO 69 <211> LENGTH: 80 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 69tttcagggac cctccaggct ttactaagcc tcccccagac tccaccagca ttacttcaga 60gtggacacct gtagctgttg 80 <210> SEQ ID NO 70 <211> LENGTH: 80 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 70 ccttcttgca cagtaataca tagccgtgtc ctcagatctcagactgctca tttgcagata 60 cagggtgttc ttggcattgt 80 <210> SEQ ID NO 71<211> LENGTH: 70 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: /Note = Synthetic Construct <400> SEQUENCE: 71 gaccgatgggcccttggtgg aggctgagga gactgtgaga gtggtccctt ggccccagta 60 gtccgtcgta 70<210> SEQ ID NO 72 <211> LENGTH: 212 <212> TYPE: PRT <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 72Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 1015 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 2530 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 4045 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 7075 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Arg Thr 8590 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyThr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg GluAla Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly AsnSer Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr GluLys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu SerSer Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu 210 <210> SEQ IDNO 73 <211> LENGTH: 213 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 73 AspIle Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Arg Thr 85 9095 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> SEQ IDNO 74 <211> LENGTH: 213 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 74 AspIle Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr 85 9095 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> SEQ IDNO 75 <211> LENGTH: 213 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 75 AspIle Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580 Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr 85 9095 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> SEQ IDNO 76 <211> LENGTH: 213 <212> TYPE: PRT <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 76 AspIle Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 7580 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr 85 9095 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Val Ala Ala Pro 100105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Gly Thr TyrSer Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> SEQ IDNO 77 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 77gtcccccaca gatgcagaca aagaacttgg agattgggtc atctgaatgt caccagtgga 60<210> SEQ ID NO 78 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 78atctagttct cagagatgga gacagacaca atcctgctat gggtgctgct gctctgggtt 60ccagg 65 <210> SEQ ID NO 79 <211> LENGTH: 69 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 79 cagcacccat agcaggattg tgtctgtctc catctctgag aactagatgagaggatgctt 60 cttaagctt 69 <210> SEQ ID NO 80 <211> LENGTH: 67 <212>TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 80 ctccactggt gacattgtga tgacccaatc tccaagttctttgtctgcat ctgtggggga 60 cagggtc 67 <210> SEQ ID NO 81 <211> LENGTH: 54<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 81 acttggagat tgggtcatca caatgtcacc agtggagcctggaacccaga gcag 54 <210> SEQ ID NO 82 <211> LENGTH: 67 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 82 accatcacct gcaaggccag tcaggatgtg ggtactgctgtagcctggta ccaacagaaa 60 ccaggaa 67 <210> SEQ ID NO 83 <211> LENGTH: 72<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 83 tacagcagta cccacatcct gactggcctt gcaggtgatggtgaccctgt cccccacaga 60 tgcagacaaa ga 72 <210> SEQ ID NO 84 <211>LENGTH: 71 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:/Note = Synthetic Construct <400> SEQUENCE: 84 aagcacccaa actcctcatctattgggcat ccacccggca cactggggtc ccagataggt 60 ttacaggcag t 71 <210> SEQID NO 85 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: artificialsequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 85cccagtgtgc cgggtggatg cccaatagat gaggagtttg ggtgcttttc ctggtttctg 60ttggtaccag gc 72 <210> SEQ ID NO 86 <211> LENGTH: 63 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 86 gggtctggga cagacttcac cctcaccatc tctagtctgcagccggagga ttttgcaacc 60 tat 63 <210> SEQ ID NO 87 <211> LENGTH: 60<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 87 actagagatg gtgagggtga agtctgtccc agacccactgcctgtaaacc tatctgggac 60 <210> SEQ ID NO 88 <211> LENGTH: 57 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 88 tactgtcagc aatatagcag ctatcggacg ttcggtcaaggcaccaaggt ggaaatc 57 <210> SEQ ID NO 89 <211> LENGTH: 57 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 89 cgtccgatag ctgctatatt gctgacagta ataggttgcaaaatcctccg gctgcac 57 <210> SEQ ID NO 90 <211> LENGTH: 51 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 90 aaacggactg tggctgcacc atctgtcttc atcttcccgccatctgatga g 51 <210> SEQ ID NO 91 <211> LENGTH: 63 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 91 gaagatgaag acagatggtg cagccacagt ccgtttgatttccaccttgg tgccttgacc 60 gaa 63 <210> SEQ ID NO 92 <211> LENGTH: 57<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 92 ttctgtcagc aatatagcag ctatcggacg ttcggtcaaggcaccaaggt ggaaatc 57 <210> SEQ ID NO 93 <211> LENGTH: 57 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 93 cgtccgatag ctgctatatt gctgacagaa ataggttgcaaaatcctccg gctgcag 57 <210> SEQ ID NO 94 <211> LENGTH: 63 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 94 gggtctggga cagacttcac cctcaccatc tctagtctgcagccggagga ttttgcagat 60 tat 63 <210> SEQ ID NO 95 <211> LENGTH: 57<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 95 ttctgtcagc aatatagcag ctatcggacg ttcggtggaggcaccaaggt ggaaatc 57 <210> SEQ ID NO 96 <211> LENGTH: 57 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 96 cgtccgatag ctgctatatt gctgacagaa ataatctgcaaaatcctccg gctgcag 57 <210> SEQ ID NO 97 <211> LENGTH: 51 <212> TYPE:DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 97 tgatgtggac atgaatttgt gagactgggt catcacaatgtcaccagtgg a 51 <210> SEQ ID NO 98 <211> LENGTH: 51 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 98 tgggttccag gctccactgg tgacattgtg atgacccagtctcacaaatt c 51 <210> SEQ ID NO 99 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 99 aagacagatg gtgcagccac agcccgtttg atttccagcttggtgcctc 49 <210> SEQ ID NO 100 <211> LENGTH: 45 <212> TYPE: DNA <213>ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: /Note = Synthetic Construct <400>SEQUENCE: 100 aagctggaaa tcaaacgggc tgtggctgca ccatctgtct tcatc 45 <210>SEQ ID NO 101 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: /Note = Synthetic Construct <400> SEQUENCE: 101agatttcaac tgctcatcag atggcgggaa 30 <210> SEQ ID NO 102 <211> LENGTH: 63<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: /Note = SyntheticConstruct <400> SEQUENCE: 102 gaagatgaag acagatggtg cagccacagtccgtttgatt tccaccttgg tgcctccacc 60 gaa 63

What is claimed is:
 1. A purified antibody which specifically binds aTRAIL receptor DR4, wherein said antibody, in its soluble form, has invivo and in vitro apoptosis-inducing activity in target cells expressingDR4.
 2. The purified antibody of claim 1, wherein the apoptosis-inducingactivity is characterized by less than 60% target cell viability atantibody concentrations of less than 30 μg/ml.
 3. The purified antibodyof claim 1, wherein said antibody is a monoclonal antibody.
 4. Thepurified antibody of claim 1, wherein the DR4 is human DR4.
 5. Thepurified antibody of claim 1, wherein the target cell is a cancer cell.6. The purified antibody of claim 1, wherein the target cell is arheumatoid arthritis synovial cell.
 7. The purified antibody of claim 1,wherein the target cell is an activated lymphocyte.
 8. A monoclonalantibody having the same epitope specificity as hybridoma 2E12 havingATCC Accession Number PTA-3798.
 9. A monoclonal antibody produced byhybridoma 2E12 having ATCC Accession Number PTA-3798.
 10. The antibodyof claim 1, wherein said antibody is humanized.
 11. A method ofselectively inducing apoptosis in target cells expressing DR4,comprising the step of contacting the target cells with a therapeuticquantity of the antibody of claim
 1. 12. The method of claim 11, whereinthe contacting is in vivo.
 13. The method of claim 11, wherein thecontacting is in vitro.
 14. The method of claim 11, wherein the targetcell is a cancer cell.
 15. The method of claim 11, wherein the targetcell is a rheumatoid arthritis synovial cell.
 16. The method of claim11, wherein the target cell is an activated lymphocyte.
 17. The methodof claim 11, wherein the target cell is a virally infected cell.
 18. Themethod of claim 11, further comprising contacting the target cells witha therapeutic quantity of an antibody which specifically binds a TRAILreceptor DR5, wherein said second antibody, in its soluble form, has invivo and in vitro apoptosis-inducing activity in target cells expressingDR5.
 19. The method of claim 11, further comprising contacting thetarget cells with a therapeutic agent.
 20. The method of claim 19,wherein the therapeutic agent is a chemotherapeutic agent.
 21. Themethod of claim 19, wherein the therapeutic agent is selected from thegroup consisting of bleomycin, carboplatin, chlorambucil, cisplatin,colchicine, cyclophosphamide, daunorubicin, actinomycin,diethylstilbestrol doxorubicin, etoposide, 5-fluorouracil, floxuridine,melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide,6-thioguanine, vincristine and vinblastine.
 22. The method of claim 19,wherein the therapeutic agent is doxorubicin.
 23. The method of claim19, wherein the therapeutic agent is paclitaxel.
 24. The method of claim19, wherein the therapeutic agent is methotrexate.
 25. The method ofclaim 11, further comprising irradiating the target cells.
 26. Themethod of claim 18, further comprising contacting the target cells witha therapeutic agent.
 27. The method of claim 26, wherein the therapeuticagent is a chemotherapeutic agent.
 28. The method of claim 26, whereinthe therapeutic agent is selected from the group consisting ofbleomycin, carboplatin, chlorambucil, cisplatin, colchicine,cyclophosphamide, daunorubicin, actinomycin, diethylstilbestroldoxorubicin, etoposide, 5-fluorouracil, floxuridine, melphalan,methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine,vincristine and vinblastine.
 29. The method of claim 26, wherein thetherapeutic agent is doxorubicin.
 30. The method of claim 26, whereinthe therapeutic agent is paclitaxel.
 31. The method of claim 26, whereinthe therapeutic agent is methotrexate.
 32. The method of claim 18,further comprising irradiating the target cells.
 33. The method of claim19, further comprising irradiating the target cells.
 34. A method ofselectively inducing apoptosis in target cells expressing DR4,comprising the step of contacting the target cells with a therapeuticquantity of the antibody of claim
 10. 35. A method of inhibitingproliferation of target cells expressing DR4, comprising the step ofcontacting the cells with a therapeutic quantity of the antibody ofclaim
 1. 36. The method of claim 35, wherein the contacting is in vivo.37. The method of claim 35, wherein the contacting is in vitro.
 38. Themethod of claim 35, wherein the target cell is a cancer cell.
 39. Themethod of claim 35, wherein the target cell is a rheumatoid arthritissynovial cell.
 40. The method of claim 35, wherein the target cell is anactivated lymphocyte.
 41. The method of claim 35, further comprisingcontacting the target cells with a therapeutic quantity of an antibodywhich specifically binds a TRAIL receptor DR5, wherein said secondantibody, in its soluble form, has in vivo and in vitroapoptosis-inducing activity in target cells expressing DR5.
 42. Themethod of claim 35, further comprising contacting the target cells witha therapeutic agent.
 43. The method of claim 42, wherein the therapeuticagent is a chemotherapeutic agent.
 44. The method of claim 42, whereinthe therapeutic agent is selected from the group consisting ofbleomycin, carboplatin, chlorambucil, cisplatin, colchicine,cyclophosphamide, daunorubicin, actinomycin, diethylstilbestroldoxorubicin, etoposide, 5-fluorouracil, floxuridine, melphalan,methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine,vincristine and vinblastine.
 45. The method of claim 42, wherein thetherapeutic agent is doxorubicin.
 46. The method of claim 42, whereinthe therapeutic agent is paclitaxel.
 47. The method of claim 42, whereinthe therapeutic agent is methotrexate.
 48. The method of claim 35,further comprising irradiating the target cells.
 49. The method of claim41, further comprising contacting the target cells with a therapeuticagent.
 50. The method of claim 49, wherein the therapeutic agent is achemotherapeutic agent.
 51. The method of claim 49, wherein thetherapeutic agent is selected from the group consisting of bleomycin,carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide,daunorubicin, actinomycin, diethylstilbestrol doxorubicin, etoposide,5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin,6-mercaptopurine, teniposide, 6-thioguanine, vincristine andvinblastine.
 52. The method of claim 49, wherein the therapeutic agentis doxorubicin.
 53. The method of claim 49, wherein the therapeuticagent is paclitaxel.
 54. The method of claim 49, wherein the therapeuticagent is methotrexate.
 55. The method of claim 41 further comprisingirradiating the target cells.
 56. The method of claim 42, furthercomprising irradiating the target cells.
 57. A method of inhibitingproliferation of target cells expressing DR4, comprising the step ofcontacting the target cells with a therapeutic quantity of the antibodyof claim
 10. 58. A composition comprising a therapeutic amount of theantibody of claim 1 and a pharmaceutically acceptable carrier.
 59. Acomposition comprising a therapeutic amount of the antibody of claim 10and a pharmaceutically acceptable carrier.
 60. A method of treating asubject with cancer, comprising administering to the subject atherapeutic amount of the antibody of claim 1, wherein the therapeuticamount of the antibody selectively induces apoptosis of cancer cells inthe subject.
 61. The method of claim 60, further comprisingadministering to the subject a therapeutic quantity of an antibody whichspecifically binds a TRAIL receptor DR5, wherein said second antibody,in its soluble form, has in vivo and in vitro apoptosis-inducingactivity in malignant cells expressing DR5.
 62. The method of claim 60,further comprising administering to the subject a therapeutic agent. 63.The method of claim 62, wherein the therapeutic agent is achemotherapeutic agent.
 64. The method of claim 62, wherein thetherapeutic agent is selected from the group consisting of bleomycin,carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide,daunorubicin, actinomycin, diethylstilbestrol doxorubicin, etoposide,5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin,6-mercaptopurine, teniposide, 6-thioguanine, vincristine andvinblastine.
 65. The method of claim 62, wherein the therapeutic agentis doxorubicin.
 66. The method of claim 62, wherein the therapeuticagent is methotrexate.
 67. The method of claim 60, further comprisingadministering to the subject irradiation treatment.
 68. The method ofclaim 61, further comprising administering to the subject a therapeuticagent.
 69. The method of claim 68, wherein the therapeutic agent is achemotherapeutic agent.
 70. The method of claim 68, wherein thetherapeutic agent is selected from the group consisting of bleomycin,carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide,daunorubicin, actinomycin, diethylstilbestrol doxorubicin, etoposide,5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin,6-mercaptopurine, teniposide, 6-thioguanine, vincristine andvinblastine.
 71. The method of claim 68, wherein the therapeutic agentis doxorubicin.
 72. The method of claim 68, wherein the therapeuticagent is paclitaxel.
 73. The method of claim 69, wherein the therapeuticagent is methotrexate.
 74. The method of claim 61, further comprisingadministering to the subject irradiation treatment.
 75. The method ofclaim 62, further comprising administering to the subject irradiationtherapy.
 76. A method of treating a subject with cancer, comprisingadministering to the subject a therapeutic amount of the antibody ofclaim 10, wherein the therapeutic amount of the antibody selectivelyinduces apoptosis of cancer cells in the subject.
 77. A method oftreating a subject having an inflammatory or autoimmune disease,comprising administering to the subject a therapeutic amount of theantibody of claim 1, wherein the therapeutic amount of the antibodyselectively induces apoptosis of target cells having DR4 receptors. 78.The method of claim 77, wherein the target cells are activated immunecells.
 79. The method of claim 78, wherein the activated immune cellsare activated lymphocytes.
 80. The method of claim 77, wherein thetarget cells are rheumatoid arthritis synovial cells.
 81. The method ofclaim 77, further comprising administering to the subject a therapeuticamount of a second antibody that induces apoptosis of the target cells.82. The method of claim 77, further comprising administering to thesubject a therapeutic amount of a therapeutic agent used in thetreatment of inflammatory disease or autoimmune disease.
 83. The methodof claim 81, further comprising administering to the subject atherapeutic amount of a therapeutic agent used in the treatment ofinflammatory disease or autoimmune disease.
 84. A method of treating asubject having an inflammatory or autoimmune disease, comprisingadministering to the subject a therapeutic amount of the antibody ofclaim 10, wherein the therapeutic amount of the antibody selectivelyinduces apoptosis of target cells having DR4 receptors.
 85. An isolatednucleic acid, comprising a nucleotide sequence that encodes a heavychain immunoglobulin of an antibody capable of binding TRAIL receptorDR4 and capable of inducing apoptosis of a cell with DR4 receptors. 86.The isolated nucleic acid of claim 85, wherein the nucleotide sequenceencodes the heavy chain immunoglobulin expressed by hybridoma 2E12having ATCC Accession Number PTA-3798.
 87. The isolated nucleic acid ofclaim 85, wherein the nucleotide sequence encodes the heavy chainimmunoglobulin expressed by hybridoma 2E12 having ATCC Accession NumberPTA-3798 with one or more conservative amino acid substitutions.
 88. Theisolated nucleic acid of claim 85, wherein the encoded heavy chain ishumanized.
 89. A vector comprising the nucleic acid of claim 88 and aregulatory element operably linked to said nucleic acid.
 90. A culturedcell comprising the vector of claim
 89. 91. An isolated nucleic acid,comprising a nucleotide sequence that encodes a light chainimmunoglobulin of an antibody capable of binding TRAIL receptor DR4 andcapable of inducing apoptosis of a cell with DR4 receptors.
 92. Theisolated nucleic acid of claim 91, wherein the nucleotide sequenceencodes the light chain immunoglobulin expressed by hybridoma 2E12having ATCC Accession Number PTA-3798.
 93. The isolated nucleic acid ofclaim 91, wherein the nucleotide sequence encodes the light chainimmunoglobulin expressed by hybridoma 2E12 having ATCC Accession NumberPTA-3798 with one or more conservative amino acid substitutions.
 94. Theisolated nucleic acid of claim 91, wherein the encoded light chain ishumanized.
 95. A vector comprising the nucleic acid of claim 94 and aregulatory element operably linked to said nucleic acid.
 96. A culturedcell comprising the vector of claim
 95. 97. A purified polypeptidecomprising an amino acid sequence of a heavy chain immunoglobulin of anantibody capable of binding TRAIL receptor DR4 and capable of inducingapoptosis of a cell with DR4 receptors.
 98. The purified polypeptide ofclaim 97, wherein the amino acid sequence is the amino acid sequence ofthe heavy chain immunoglobulin expressed by hybridoma 2E12 having ATCCAccession Number PTA-3798.
 99. The purified polypeptide of claim 97,wherein the amino acid sequence is the amino acid of the heavy chainimmunoglobulin expressed by hybridoma 2E12 having ATCC Accession NumberPTA-3798 with one or more conservative amino acid substitutions. 100.The purified polypeptide of claim 97, wherein the heavy chainimmunoglobulin is humanized.
 101. A purified polypeptide comprising anamino acid sequence of a light chain immunoglobulin of an antibodycapable of binding TRAIL receptor DR4 and capable of inducing apoptosisof a cell with DR4 receptors.
 102. The purified polypeptide of claim101, wherein the amino acid sequence is the amino acid sequence of thelight chain immunoglobulin expressed by hybridoma 2E12 having ATCCAccession Number PTA-3798.
 103. The purified polypeptide of claim 101,wherein the amino acid sequence is the amino acid of the light chainimmunoglobulin expressed by hybridoma 2E12 having ATCC Accession NumberPTA-3798 with one or more conservative amino acid substitutions. 104.The purified polypeptide of claim 101, wherein the light chainimmunoglobulin is humanized.